Plant transformation

ABSTRACT

Plant cells and related systems, methods, and compositions for improving the capacity of the plant cells to regenerate embryogenic plant tissues, plant organs, and whole plants are provided. Such plant cells and related systems, methods, and compositions provide for increased expression of the endogenous morphoregulators BABYBOOM (ODP2) and/or WUSCHEL2 (WUS2).

REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. Ser. No. 16/844,438, filed Apr. 9, 2020, which claims the benefit of priority to U.S. Provisional Patent Application No. 62/833,150, filed Apr. 12, 2019, which are incorporated herein by reference in their entireties.

INCORPORATION OF SEQUENCE LISTING

The sequence listing contained in the file named “63200_195755_SEQLISTING_ST25.txt”, which is 385,024 bytes measured in Windows, which was created on Apr. 9, 2020 and electronically filed via EFS-Web on Apr. 9, 2020, is incorporated herein by reference in its entirety.

BACKGROUND

To overcome recalcitrance of corn to genetic transformation, transgenes encoding morphoregulators BABYBOOM (ODP2) and/or WUSCHEL2 (WUS2) have been transiently expressed in corn cells to stimulate somatic embryogenesis (Lowe et al. 2016, Lowe et al. 2018). Such somatic embryogenesis promotes formation of Type II embryogenic callus from which new shoots can be generated, allowing maize genotype independent genetic transformation and making previously recalcitrant tissues accessible to transformation. The expression of BABYBOOM (ODP2) and/or WUSCHEL2 (WUS2) has also been shown to promote embryogenesis in sorghum (Mookkan et al. 2017), wheat, and a variety of other plants including cotton (US Patent Appl. Pub. No. 20170342431; U.S. Pat. No. 7,256,322; also reviewed by Nagle et al., 2018).

It is not yet clear how BABYBOOM and WUSCHEL trigger this effect. BABYBOOM is known to induce somatic embryogenesis, the biology of which is beginning to be understood (Horstman et al. 2017; Jha and Kumar, 2019). WUSCHEL biology has been investigated primarily in Arabidopsis (Rodriguez et al. 2016; Schoof et al. 2000; Mayer et al. 1998; Laux et al. 1996). It has a major role in stem cell maintenance.

The genomic insertion of morphoregulator-encoding transgenes is not a universal agricultural biotechnology solution, and also stipulates market access consequences. Commercialization of transgenic plants, and their progeny, is restricted by country-specific regulations. Genomic insertion of transgenes can facilitate the introduction of gene editing reagents, for example when coupled with transgenes encoding CRISPR-Cas9 (Soda, Verma, and Giri 2017; W. Wang et al. 2018). Hence, transgenes often need to be segregated away from edited progeny which can require multiple crosses and lengthen development/production timelines and costs. Transgenes do enable the use of selectable markers which enrich for edited tissue, greatly improving editing efficiency. Due to this enrichment step transgenesis remains the primary method for producing edited plants, however several groups are experimenting with alternative selection tools (Zhang et al. 2016; Hamada et al. 2018).

SUMMARY

Disclosed herein are plant cells wherein expression of an endogenous ODP2 polypeptide and/or expression of an endogenous WUS2 polypeptide is transiently increased in comparison to the expression of the endogenous ODP2 and/or the endogenous WUS2 polypeptides in a control plant cell, and wherein the plant cell can form a regenerable plant structure. Also disclosed are tissue cultures of such plant cells and related methods wherein the cells are used to obtain genetically edited or genetically transformed regenerable plant structures (e.g., a somatic embryo, embryogenic callus, somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots) or plants. In certain embodiments, the expression of the endogenous ODP2 polypeptide and/or the endogenous WUS2 polypeptide is transiently increased in the plant cell with at least one exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or with at least one exogenous gene transcription agent that stimulates transcription of the endogenous WUS2 gene. Such plant cells include both monocot (e.g., maize, wheat, sorghum, and rice) plant cells and dicot plant cells (e.g., Brassica sp., cotton, and soybean). Also provided are maize plant cells comprising at least one exogenous gene transcription agent that stimulates transcription of the endogenous WUS2 gene, wherein expression of the endogenous WUS2 polypeptide is increased in comparison to the expression of the endogenous WUS2 polypeptide in a control maize plant cell, wherein the endogenous WUS2 polypeptide is encoded by an endogenous polynucleotide that is operably linked to an endogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variant thereof, wherein the exogenous gene transcription agent(s) bind to DNA sequences in the endogenous maize WUS2 promoter corresponding to residues 100 to 225 of SEQ ID NO:4, and wherein the maize plant cells can form a regenerable maize plant structure.

Methods provided herein include methods of producing a regenerable plant structure, comprising introducing into the plant cell at least one exogenous gene transcription agent which transiently increases expression of an endogenous ODP2 polypeptide and/or at least one exogenous gene transcription agent which increases expression of an endogenous WUS2 polypeptide, wherein the expression is increased in comparison to the expression of the endogenous ODP2 and/or the endogenous WUS2 polypeptides in a control plant cell; and culturing the plant cell to produce the regenerable plant structure. In certain embodiments of the methods, the exogenous gene transcription agent comprises: (i) a domain or complex which binds to the promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 gene or to the promoter or 5′ UTR of the endogenous WUS2 gene; and (ii) a transcription activation domain, wherein the transcription activation domain is operably linked or operably associated with the domain or complex. Such methods can be applied to plant cells that include both monocot (e.g., maize, wheat, sorghum, and rice) plant cells and dicot plant cells (e.g., Brassica sp., cotton, and soybean). Also provided are methods of producing a regenerable maize plant structure, comprising: (i) introducing into a maize plant cell at least one exogenous gene transcription agent which transiently increases expression of an endogenous WUS2 polypeptide, wherein the expression is increased in comparison to the expression of the endogenous WUS2 polypeptide in a control maize plant cell, wherein the endogenous WUS2 polypeptide is encoded by an endogenous polynucleotide that is operably linked to an endogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variant thereof, and wherein the exogenous gene transcription agent(s) bind to DNA sequences in the endogenous maize WUS2 promoter corresponding to residues 100 to 225 of SEQ ID NO:4; and, (ii) culturing the maize plant cell to produce a regenerable maize plant structure.

DETAILED DESCRIPTION

Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5′ to 3′ direction. Nucleic acid sequences may be provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as well as necessarily defines the exact complements, as is known to one of ordinary skill in the art. Where a term is provided in the singular, the inventors also contemplate embodiments described by the plural of that term.

The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variety, or isolate of a given organism.

The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the terms “correspond,” “corresponding,” and the like, when used in the context of an nucleotide position, mutation, and/or substitution in any given polynucleotide (e.g., an allelic variant of SEQ ID NO: 4) with respect to the reference polynucleotide sequence (e.g., SEQ ID NOs: 4, 101, 102, residues 130-210 of SEQ ID NO: 4) all refer to the position of the polynucleotide residue in the given sequence that has identity to the residue in the reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide sequence using a pairwise alignment algorithm (e.g., CLUSTAL O 1.2.4 with default parameters).

As used herein, the terms “Cpf1” and “Cas12a” are used interchangeably herein to refer to the same RNA directed nuclease.

The phrases “expression of an endogenous ODP2 polypeptide,” “expression of an endogenous WUS2 polypeptide,” “expression of the endogenous ODP2 polypeptide,” and “expression of the endogenous WUS2 polypeptide” refer to the expression of an ODP2 polypeptide or WUS2 polypeptide respectively encoded by an endogenous ODP2 gene or endogenous WUS2 gene in a plant genome.

As used herein, the phrase “genome altering reagent’ refers to any molecule or set of molecules that can result in either the site-specific or non-site specific insertion of an exogenous nucleic acid molecule into the genome or a site-specific or non-site specific insertion, deletion, and/or substitution of one or more nucleotide residues in the genome. A genome altering reagent can comprise a transgene, a vector comprising a transgene, a genome editing molecule(s), and/or polynucleotides encoding the genome editing molecule(s).

As used herein, the phrase “gene-editing” includes genome modification by homology directed repair (HDR), base editing, and non-homologous end-joining (NHEJ) mechanisms. Such gene-editing includes embodiments where a site specific nuclease and a donor template are provided.

As used herein, an “exogenous” agent or molecule refers to any agent or molecule from an external source that is provided to or introduced into a system, composition, plant cell culture, reaction system, or plant cell. In certain embodiments, the exogenous agent (e.g., polynucleotide, protein, or compound) from the external source can be an agent that is also found in a plant cell. In certain embodiments, the exogenous agent (e.g., polynucleotide, protein, or compound) from the external source can be an agent that is heterologous to the plant cell.

As used herein, a “heterologous” agent or molecule refers: (i) to any agent or molecule that is not found in a wild-type, untreated, or naturally occurring composition or plant cell; and/or (ii) to a polynucleotide or peptide sequence located in, e.g., a genome or a vector, in a context other than that in which the sequence occurs in nature. For example, a promoter that is operably linked to a gene other than the gene that the promoter is operably linked to in nature is a heterologous promoter.

The phrase “improved plant cell regenerative potential” as used herein refers to the ability of a given plant cell to form a somatic embryo, embryogenic callus, a somatic meristem, organogenic callus, a shoot, or a shoot further comprising roots in comparison to a control plant cell.

As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.

As used herein, the term “overproduced” where used herein with regards to various agents refers to providing the agent in an amount that is increased in comparison to the amount found in an untreated plant cell or plant.

As used herein, the phrase “plant cell” can refer either a plant cell having a plant cell wall or to a plant cell protoplast lacking a plant cell wall.

The term “polynucleotide” where used herein is a nucleic acid molecule containing two (2) or more nucleotide residues. Polynucleotides are generally described as single- or double-stranded. Where a polynucleotide contains double-stranded regions formed by intra- or intermolecular hybridization, the length of each double-stranded region is conveniently described in terms of the number of base pairs. Embodiments of the systems, methods, and compositions provided herein can employ or include: (i) one or more polynucleotides of 2 to 25 residues in length, one or more polynucleotides of more than 26 residues in length, or a mixture of both. Polynucleotides can comprise single- or double-stranded RNA, single- or double-stranded DNA, double-stranded DNA/RNA hybrids, chemically modified analogues thereof, or a mixture thereof. In certain embodiments, a polynucleotide can include a combination of ribonucleotides and deoxyribonucleotides (e.g., synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides), or can include non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides (see, e.g., Verma and Eckstein (1998) Annu. Rev. Biochem., 67:99-134). Chemically modified nucleotides that can be used in the polynucleotides provided herein include: (i) phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications of the phosphodiester backbone; (ii) nucleosides comprising modified bases and/or modified sugars; and/or (iii) detectable labels including a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels) or other label (e.g., biotin or an isotope). Polynucleotides provided or used herein also include modified nucleic acids, particularly modified RNAs, which are disclosed in U.S. Pat. No. 9,464,124, which is incorporated herein by reference in its entirety.

As used herein the term “synergistic” refers to an effect of combining at least two factors that exceeds the sum of the effects obtained when the factors are not combined.

As used herein, the phrase “target plant gene” can refer to either a gene located in the plant genome that is to be modified by gene editing molecules provided in a system, method, composition and/or plant cell provided herein or alternatively to a plant gene located in the plant genome that is targeted for increased expression (e.g., an ODP2 and/or an WUS2 gene). Embodiments of target plant genes include (protein-)coding sequence, non-coding sequence, and combinations of coding and non-coding sequences. Modifications of a target plant gene include nucleotide substitutions, insertions, and/or deletions in one or more elements of a plant gene that include a transcriptional enhancer or promoter, a 5′ or 3′ untranslated region, a mature or precursor RNA coding sequence, an intron, a splice donor and/or acceptor, a protein coding sequence, a polyadenylation site, and/or a transcriptional terminator. In certain embodiments, all copies or all alleles of a given target gene in a diploid or polyploid plant cell are modified to provide homozygosity of the modified target gene in the plant cell. In embodiments, where a desired trait is conferred by a loss-of-function mutation that is introduced into the target gene by gene editing, a plant cell, population of plant cells, plant, or seed is homozygous for a modified target gene with the loss-of-function mutation. In other embodiments, only a subset of the copies or alleles of a given target gene are modified to provide heterozygosity of the modified target gene in the plant cell. In certain embodiments where a desired trait is conferred by a dominant mutation that is introduced into the target gene by gene editing, a plant cell, population of plant cells, plant, or seed is heterozygous for a modified target gene with the dominant mutation. Traits imparted by such modifications to certain plant target genes include improved yield, resistance to insects, fungi, bacterial pathogens, and/or nematodes, herbicide tolerance, abiotic stress tolerance (e.g., drought, cold, salt, and/or heat tolerance), protein quantity and/or quality, starch quantity and/or quality, lipid quantity and/or quality, secondary metabolite quantity and/or quality, and the like, all in comparison to a control plant that lacks the modification. The plant having a genome modified by gene editing molecules provided in a system, method, composition and/or plant cell provided herein differs from a plant having a genome modified by traditional breeding (i.e., crossing of a male parent plant and a female parent plant), where unwanted and random exchange of genomic regions as well as random mitotically or meiotically generated genetic and epigenetic changes in the genome typically occurs during the cross and are then found in the progeny plants. Thus, in embodiments of the plant (or plant cell) with a modified genome, the modified genome is more than 99.9% identical to the original (unmodified) genome. In embodiments, the modified genome is devoid of random mitotically or meiotically generated genetic or epigenetic changes relative to the original (unmodified) genome. In embodiments, the modified genome includes a difference of epigenetic changes in less than 0.01% of the genome relative to the original (unmodified) genome. In embodiments, the modified genome includes: (a) a difference of DNA methylation in less than 0.01% of the genome, relative to the original (unmodified) genome; or (b) a difference of DNA methylation in less than 0.005% of the genome, relative to the original (unmodified) genome; or (c) a difference of DNA methylation in less than 0.001% of the genome, relative to the original (unmodified) genome. In embodiments, the gene of interest is located on a chromosome in the plant cell, and the modified genome includes: (a) a difference of DNA methylation in less than 0.01% of the portion of the genome that is contained within the chromosome containing the gene of interest, relative to the original (unmodified) genome; or (b) a difference of DNA methylation in less than 0.005% of the portion of the genome that is contained within the chromosome containing the gene of interest, relative to the original (unmodified) genome; or (c) a difference of DNA methylation in less than 0.001% of the portion of the genome that is contained within the chromosome containing the gene of interest, relative to the original (unmodified) genome. In embodiments, the modified genome has not more unintended changes in comparison to the original (unmodified) genome than 1×10{circumflex over ( )}-8 mutations per base pair per replication. In certain embodiments, the modified genome has not more unintended changes than would occur at the natural mutation rate. Natural mutation rates can be determined empirically or are as described in the literature (Lynch, M., 2010; Clark et al., 2005).

To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

Plant cells and related systems, methods, and compositions that provide for improved plant cell regenerative potential in comparison to control plant cells are provided herein. In certain embodiments, improved plant cell regenerative potential is provided by transiently increasing the expression of an ODP2 polypeptide and/or WUS2 polypeptide respectively encoded by an endogenous ODP2 gene or endogenous WUS2 gene in a plant genome in the plant cells in comparison to a control plant cell. Such transient expression of the endogenous WUS2 and/or ODP2 genes can provide for desired improvements in the regenerative capacity of the plant cell while avoiding undesired effects of increasing expression of WUS2 and/or ODP2 in partially or fully regenerated plant structures, tissues, organs, and plants. Transient expression of the endogenous ODP2 and/or WUS2 genes can be for a period of time and/or in an amount sufficient to result in improved regenerative potential in comparison to a control plant cell. In certain embodiments, the transient increase in the expression of the endogenous ODP2 polypeptide and/or expression of the endogenous WUS2 polypeptide is for a period of about 1, 2, 4, 8, 12, 16, 20, 24, 30, or 36 hours to about 72, 96, 120, 144, 168, 192, 276, or 336 hours. In certain embodiments, the transient increase in the expression of the endogenous ODP2 polypeptide and/or expression of the endogenous WUS2 polypeptide is for a period of about 2, 4, 8, 12, or 16 hours to about 18, 20, 24, 30 or 36 hours. In certain embodiments, the transient increase in the expression of the endogenous ODP2 polypeptide and/or expression of the endogenous WUS2 polypeptide is for a period of about 18, 20, 24, 30 or 36 hours to about 60, 80, 100, 120, 168, or 192 hours. Such transient increases in expression of the endogenous ODP2 and/or WUS2 genes can be measured by methods whereby accumulated ODP2 and/or WUS2 gene products including mRNAs and/or proteins are measured. Useful methods of measuring ODP2 and/or WUS2 mRNAs include quantitative reverse transcriptase Polymerase Chain Reaction (qRT-PCR)-based and/or any hybridization-based assay. Useful methods for quantitating ODP2 and/or WUS2 include immunoassays (e.g., ELISAs, RIAs) and/or mass spectrometry-based methods. In certain embodiments, expression of endogenous ODP2 and/or WUS2 gene products including mRNAs and/or proteins are transiently increased by at least 1.5-, 2-, 3-, 5-, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold in comparison to the corresponding endogenous ODP2 and/or WUS2 gene products in a control plant cell. In certain embodiments, expression of endogenous ODP2 and/or WUS2 gene products including mRNAs and/or proteins are transiently increased by at least 1.5-, 2-, or 3-fold to about 4-, 5-, 10-, 15-, 20-, 50-, 100-, 500-f, or 1000-fold in comparison to the corresponding endogenous ODP2 and/or WUS2 gene products in a control plant cell.

Endogenous ODP2 genes in plants that can be targeted for increased expression by methods provided herein include the endogenous ODP2 genes of both monocot and dicot plants. Such endogenous ODP2 genes include the ODP2 genes that encode ODP2 peptides disclosed in US Patent Application Publication Nos. 20190017061 and 20170121722, which are specifically incorporated herein by reference in their entireties with respect to such disclosure of such ODP2 genes and peptides. Endogenous ODP2 genes targeted for increased expression can encode ODP2 peptides that comprise APETALA2 (AP2) DNA binding motifs, and amino acid variants thereof. In certain embodiments, the plant cell is a maize plant cell and the endogenous ODP2 gene targeted for increased expression encodes a ODP2 polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, or 99% amino acid sequence identity across the entire length of SEQ ID NO:1. In certain embodiments, the plant cell is a maize plant cell and the endogenous ODP2 gene targeted for increased expression of the ODP2 polypeptide is the endogenous maize ODP2 gene located on maize chromosome 3. In certain embodiments, the plant cell is a maize plant cell and the endogenous ODP2 gene targeted for increased expression comprises an endogenous polynucleotide that is operably linked to an endogenous maize ODP2 promoter of SEQ ID NO:3, SEQ ID NO:71, or an allelic variant thereof.

Endogenous WUS2 genes in plants that can be targeted by methods provided herein include the endogenous WUS2 genes of both monocot and dicot plants. Such endogenous WUS2 genes include the WUS2 genes that encode WUS2 peptides disclosed in U.S. Pat. No. 7,256,322 and US Patent Application Publication No. 20170121722, which are specifically incorporated herein by reference in their entireties with respect to such disclosure of such WUS2 genes and peptides. Endogenous WUS2 genes targeted for increased expression can encode WUS2 peptides that comprise conserved homeodomain motifs such as the (E/R)TLPLFP motif (SEQ ID NO:109), the A(A/S)LEL(S/T)L motif (SEQ ID NO:110), a 25 amino acid motif located between the (E/R)TLPLFP (SEQ ID NO:109) and the A(A/S)LEL(S/T)L (SEQ ID NO:110) motifs, and amino acid variants thereof. In certain embodiments, the plant cell is a maize plant cell and the endogenous WUS2 gene targeted for increased expression encodes a WUS2 polypeptide comprising an amino acid sequence having at least 95%, 96%, 97%, or 99% amino acid sequence identity across the entire length of SEQ ID NO:2. In certain embodiments, the plant cell is a maize plant cell and the endogenous WUS2 gene targeted for increased expression of the endogenous WUS2 polypeptide the endogenous maize WUS2 gene located on maize chromosome 10. In certain embodiments, the plant cell is a maize plant cell and the endogenous WUS2 gene targeted for increased expression comprises an endogenous polynucleotide that is operably linked to an endogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variant thereof.

In certain embodiments, expression of the endogenous ODP2 and/or WUS2 gene is transiently increased by introducing at least one exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or with at least one exogenous gene transcription agent that stimulates transcription of the endogenous WUS2 gene. In certain embodiments, expression of the endogenous ODP2 and/or WUS2 gene is transiently increased by introducing an exogenous gene transcription agent that stimulates transcription of both the endogenous ODP2 gene and the endogenous WUS2 gene. In certain embodiments, additional exogenous polynucleotides encoding an ODP2 and/or WUS2 polypeptide are not provided to the cell since the exogenous transcription agents can increase the regenerative capacity of the plant cell by increasing expression of the endogenous ODP2 and/or WUS2 polypeptides encoded by the endogenous ODP2 and/or WUS2 genes. Features of the exogenous gene transcription agents that can increase expression of the endogenous ODP2 and WUS2 genes include: (a) a DNA binding domain that specifically binds a sequence within the promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 and/or WUS2 gene; (b) a transcriptional activation domain (TAD) that is operably linked or operably associated with the DNA binding domain; and, where required, (c) a nuclear localization signal (NLS) that is operably linked to the DNA binding domain. In certain embodiments, the aforementioned exogenous transcription factors are artificial transcription factors (ATFs). In certain embodiments, an exogenous gene transcription agent that stimulates transcription of both the endogenous ODP2 gene and the endogenous WUS2 gene could comprise an artificial transcription factor comprising: (a) a first DNA binding domain that specifically binds the endogenous ODP2 gene promoter or 5′ UTR, a second DNA binding domain that binds the endogenous WUS2 gene promoter or 5′ UTR; (b) a TAD that is operably linked or operably associated with the DNA binding domains, and, where required (c) an NLS that is operably linked with the DNA binding domains. In certain embodiments, the ATFs comprise one or more of the features or elements within the features are wholly synthetic (e.g., non-naturally occurring) or wherein features from heterologous proteins are combined. Specific binding to the promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 and/or WUS2 gene by the DNA binding domain can be shown by DNA binding assays. Protein-DNA binding assays that can be used include DNA electrophoretic mobility shift assays (EMSA); chromatin immunoprecipitation (ChIP)-based assays; enzyme-linked immunoassays, fluorescence-anisotropy-based assays, and surface plasmon resonance assays (Jantz and Berg, 2010). Specific DNA binding activity can also be demonstrated in competitive DNA binding assays wherein binding to the target DNA sequence is inhibited more efficiently (e.g., at lower concentrations) by the target DNA sequence located within the promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 or WUS2 gene than by an unrelated, non-target DNA sequence. In certain embodiments, the DNA binding domains and/or artificial transcription factors used herein will bind the target DNA sequence with an affinity (K_(d)) of 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less or will bind with an affinity of about 10 nM or 8 nM to about 1 nM or 0.5 nM. Other optional features of the artificial transcription factors include epitope tags that can facilitate detection and/or quantitation of expression as well as cell penetrating peptides that can facilitate entry into a target plant cell.

Transcriptional activation domains (TADs) used in the ATFs can be obtained from either naturally occurring transcription factors or can be wholly or partially synthetic. Any of an acidic, glutamine-rich, proline-rich, isoleucine-rich, and/or an alanine-rich TAD can be used (Ma, 2011). Examples of such TADs that can be used include the maize C1, the VP16, and the VP64 transcription activation domains. In certain embodiments, multiple VP64 TADs can be used (Li et al., 2018). Another example of a potent plant TAD that can be used in the ATFs provided herein is the EDLL motif that is found in AP2/ERF transcription factors (Tiwari et al., 2012). Yet another example of a potent plant TAD that can be used in the ATFs provided herein is a hybrid VP64-p65-Rta tripartite activator (VPR; SEQ ID NO: 91; Chavez et al., 2015).

Nuclear localization signals (NLS) that can be used in the ATF provided herein include monopartite and bipartite nuclear localization signals (Kosugi et al., 2009). Examples of monopartite NLS that can be used include NLS that comprise at least 4 consecutive basic amino acids such as the SV40 large T antigen NLS (PKKKRKV; SEQ ID NO:49) and another class having only three basic amino acids with a K(K/R)X(K/R) consensus sequence (SEQ ID NO:50). Examples of bipartite NLS that can be used in the ATFs provided herein include (K/R)(K/R)X₁₀₋₁₂(K/R)_(3/5) (SEQ ID NO:51) where (K/R)_(3/5) represents at least three of either lysine or arginine of five consecutive amino acids. An NLS can also comprise a plant-specific class 5 NLS having a consensus sequence of LGKR(K/R)(W/F/Y) (SEQ ID NO:52). Examples of specific NLS that can be used further include the maize opaque-2 nuclear localization signal and an extended SV40 large T antigen NLS (SEQ ID NO: 92).

In certain embodiments, the TAD and NLS elements can be operably linked to the DNA binding domain in an ATF via either a direct covalent linkage of the elements and domain or by a use of a linker peptide or flexible hinge polypeptide. Flexible hinge polypeptides include glycine-rich or glycine/serine containing peptide sequence. Such sequences can include, but are not limited to, a (Gly4)n sequence, a (Gly4Ser)n sequence of SEQ ID NO:53, a Ser(Gly4Ser)n sequence of SEQ ID NO:54, combinations thereof, and variants thereof, wherein n is a positive integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In certain embodiments, such glycine-rich or glycine/serine containing hinge peptides can also contain threonyl and/or alanyl residues for flexibility as well as polar lysyl and/or glutamyl residues. Other examples of hinge peptides that can be used include immunoglobulin hinge peptides (Vidarsson et al., 2014).

A variety of cell-penetrating peptides (CPP) can also be used in the ATF provided herein. CPPs that can be used include a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO:55); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21: 1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO:56); Transportan (e.g., GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:57)); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:58); and RQIKIWFQNRRMKWKK (SEQ ID NO:59). Exemplary CPP amino acid sequences also include

(SEQ ID NO: 60) YGRKKRRQRRR; (SEQ ID NO: 61)  RKKRRQRR; (SEQ ID NO: 62) YARAAARQARA; (SEQ ID NO: 63) THRLPRRRRRR; and (SEQ ID NO: 64) GGRRARRRRRR.

In certain embodiments, a TAD can be operably associated with a DNA binding domain via a non-covalent interaction between a protein comprising the TAD and the DNA binding peptide. In certain embodiments, such operable associations can be provided by protein domains that bind to one another (e.g., dimerization or other multimerization domains). Examples of such dimerization domains include leucine zipper structures. Such operable associates are similar to those used in yeast two-hybrid systems where interacting proteins are identified via their ability to join a TAD to a DNA binding domain (Bruckner et al., 2009). In certain embodiments, operable association can be achieved by using protein domains that interact through binding a common ligand (e.g., the iDimerize™ Regulated Transcription System that uses Dmr A, B, or C dimerization domains and ligands; Takara Bio, USA, Inc.). Such ligand-based systems have the advantage of allowing control of dimerization (and activation of the endogenous ODP2 and/or WUS2 gene expression) by ligand addition or removal.

In certain embodiments, the DNA binding domain can comprise an artificial zinc finger (AZF) DNA binding domain polypeptide which specifically binds a sequence within the promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 and/or WUS2 gene. In certain embodiments, the AZF DNA binding domain specifically binds a target DNA sequence located within the promoter or 5′ untranslated region (5′ UTR) of the endogenous maize ODP2 and/or maize WUS2 gene. Such target sequences in the endogenous maize ODP2 promoter include SEQ ID NO:6, SEQ ID NO:9, and any allelic variants thereof having one or more nucleotide insertions, deletions, and/or substitutions found in other wild-type maize genomes. AZF DNA binding domains predicted to bind the maize ODP2 promoter target sequences of SEQ ID NO:6 or SEQ ID NO:9 include the polypeptides comprising SEQ ID NO:5 and SEQ ID NO:8, respectively, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO: 5 or 8; or one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO: 5 or 8. Such target sequences in the endogenous maize WUS2 promoter include SEQ ID NO:12, SEQ ID NO:15, DNA sequences in the endogenous maize WUS2 promoter corresponding to residues 100 to 225 or 130 to 210 of SEQ ID NO:4 (or their complementary strand), SEQ ID NO:101, SEQ ID NO: 102 (in the minus or complementary strand of the dsDNA comprising SEQ ID NO: 4), and any allelic variants thereof having one or more nucleotide insertions, deletions, and/or substitutions found in other wild-type maize genomes. Such target sequences in the endogenous maize WUS2 promoter also include DNA sequences in the endogenous maize WUS2 promoter corresponding to residues 100 to 225 or 130 to 210 of SEQ ID NO:4 (or their complementary strand), and any allelic variants thereof having one or more nucleotide insertions, deletions, and/or substitutions found in other wild-type maize genomes. In certain embodiments, such allelic variants of the endogenous promoter WUS2 can have at least 80%, 85%, 90%, 95%, 97%, 98%, 98%, or 99% sequence identity to SEQ ID NO: 4, residues 100 to 225 or 130 to 210 of SEQ ID NO:4, SEQ ID NO:101, or SEQ ID NO: 102. In certain embodiments, the target sequences in the endogenous maize WUS2 promoter also include DNA sequences in the endogenous maize WUS2 promoter corresponding to: (i) residues 100, 105, 110, 115, 120, 125, 130, 132, 134, 135, 136, 137, or 138 to 155, 156, 157, 158, 160, 162, 165, or 170 of SEQ ID NO:4 (or their complementary strand); (ii) residues 171, 175, 180, 182, 183, 184, 185, 186, 187, or 188 to 202, 203, 204, 205, 206, 207, 208, 209, 210, 215, 220, or 225 of SEQ ID NO:4 (or their complementary strand); (iii) any combination of (i) and (ii); e.g., where two ATFs are used; or (iv) residues 100, 105, 110, 115, 120, 125, 130, 132, 134, 135, 136, 137, or 138 to 202, 203, 204, 205, 206, 207, 208, 209, 210, 215, 220, or 225 of SEQ ID NO:4 (or their complementary strand) of SEQ ID NO:4 (or their complementary strand). AZF-binding domains predicted to bind the maize WUS2 promoter target sequences of SEQ ID NO:12 or SEQ ID NO:15 include the polypeptides comprising SEQ ID NO:11 and SEQ ID NO:14, respectively, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:11 or 14; or one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:11 or 14. AZF DNA binding domains predicted to bind the maize WUS2 promoter target sequences comprising SEQ ID NO:101 or 102 include the polypeptides comprising SEQ ID NO: 105 or 106, respectively, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:105 or 106; or one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:105 or 106. AZF DNA binding domains predicted to bind the maize WUS2 promoter target sequences comprising SEQ ID NO:101, 102 and adjacent sequences or comprising subfragments (e.g., 9, 12, or 15 nucleotides) of SEQ ID NO:101 or 102 and adjacent sequences also include variants of SEQ ID NO:105 or 106 that further comprise additional zinc finger DNA binding motifs designed to bind the adjacent WUS2 promoter sequences. Artificial transcription factors (ATFs) comprising the aforementioned AZF DNA-binding polypeptides can further comprise operably linked nuclear localization peptides, cell-penetrating peptides, and transcription activation domains. Such ATFs predicted to bind and activate the endogenous ZmODP2 promoter include the ATFs set forth in SEQ ID NO:7 and 10, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:7 or 10; or having at one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:7 or 10. Such ATFs predicted to bind and activate the endogenous maize WUS2 (ZmWUS2) promoter include the ATFs set forth in SEQ ID NO:13 and 16, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:13 or 16; or having at one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:13 or 16. Such ATFs predicted to bind and activate the endogenous maize WUS2 (ZmWUS2) promoter include the ATFs set forth in SEQ ID NO:93 and 95, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:93 and 95; or having at one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:93 and 95. In other embodiments, target AZF DNA binding sites in the promoter or 5′UTR sequences of other endogenous plant ODP2 or WUS2 genes can be selected and AZF DNA binding domains as well as AZF transcription factors which specifically bind the target binding sites can be designed to obtain AZF transcription factors that can increase expression of other endogenous plant ODP2 or WUS2 genes. In certain embodiments, target AZF DNA binding sites can be selected based on the presence of consecutive DNA triplets that can each be recognized by zinc finger domains comprising a Cysz-Hist zinc finger motif. Target AZF DNA binding sites can be selected for the absence of overlap with sequences in non-target genes (e.g., genes other than endogenous plant ODP2 or WUS2 genes). AZF DNA binding domains, including variants of the SEQ ID NO:11, 14, 105, and 106 AZF DNA binding domains, that target the selected AZF DNA binding sites can be constructed by joining zinc finger domains. In certain embodiments, the AZF will comprise about six (6) zinc finger domains joined by canonical TGEKP (SEQ ID NO: 48) linker peptides. In certain embodiments, rules governing the design of Zn-ATFs to bind specific DNA sequences that have been published (Sera and Uranga 2002; Gersbach, Gaj, and Barbas 2014) or provided online (on the world wide web at “zincfingers.org/default2.htm” and “scripps.edu/barbas/zfdesign/zfdesignhome.php” can be applied to the design of the AZF's which bind target AZF binding sites in the ODP2 or WUS2 promoters or 5′UTR or to the construction of variants of the SEQ ID NO:11, 14, 105, and 106 AZF DNA binding domains. Features of artificial transcription factors that comprise AZF-DNA binding domains for use in activating endogenous genes in plants and other organisms that have been described in various publications (van Tol and van der Zaal 2014; Heiderscheit et al. 2018; Van Eenennaam et al. 2004; Gupta et al. 2012; Stege et al. 2002; Sanchez et al. 2006; Holmes-Davis et al. 2005; Petolino and Davies 2013) can also be used in the design of artificial transcription factors comprising AZF-DNA binding domains that recognize plant ODP2 or WUS2 promoters or 5′UTR sequences or in the construction of variants of the SEQ ID NO:11, 14, 105, and 106 AZF DNA binding domains.

In certain embodiments, the DNA binding domain can comprise an artificial transcription activator-like effector (TALE) DNA binding polypeptide (aTALE) which comprises an ‘repeat-variable di-residue’ (RVD) containing domain that specifically binds a sequence within the promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 or WUS2 gene. In certain embodiments, the TALE DNA binding polypeptide specifically binds a target DNA sequence located within the promoter or 5′ untranslated region (5′ UTR) of the endogenous maize ODP2 or maize WUS2 gene. Such target sequences in the endogenous maize ODP2 promoter include sequences within SEQ ID NO:3 or in SEQ ID NO:71, and any allelic variants thereof having one or more nucleotide insertions, deletions, and/or substitutions found in other wild-type maize genomes. TALE DNA-binding polypeptides predicted to bind the maize ODP2 (ZmODP2) promoter target sequences within SEQ ID NO:3 include the polypeptides comprising SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:81, SEQ ID NO:84, and SEQ ID NO:87, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:23, 25, 27, 81, 84, or 87; or one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO: 23, 25, 27, 81, 84, or 87. Artificial transcription factors (ATFs) comprising the aforementioned TALE DNA binding polypeptides can further comprise operably linked nuclear localization peptides, cell-penetrating peptides, and operably linked or operably associated transcription activation domains. Such ATFs predicted to bind and activate the endogenous ZmODP2 promoter include the ATFs set forth in SEQ ID NO:24, 26, 28, 82, 85, or 88, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:24, 26, 28, 82, 85, or 88; or having at one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:24, 26, 28, 82, 85, or 88. Any of the aforementioned ATFs predicted to bind the maize ODP2 promoter can be used either independently, in tandem pairs of ATFs predicted to bind at ˜100 bp intervals in ZmODP2 promoter sequences located 5′ to the ZmODP2 transcription start site, or as a set of three ATFs predicted to bind at ˜100 bp intervals in ZmODP2 promoter sequences located 5′ to the ZmODP2 transcription start site. In other embodiments, ATFs predicted to bind the maize ODP2 promoter can be used in tandem pairs of ATFs predicted to bind at ˜50 bp intervals in ZmODP2 promoter sequences located 5′ to the ZmODP2 transcription start site, or as a set of three ATFs predicted to bind at ˜50 bp intervals in ZmODP2 promoter sequences located 5′ to the ZmODP2 transcription start site. Such target sequences in the endogenous maize WUS2 promoter include sequences located within SEQ ID NO:4 and any allelic variants thereof having one or more nucleotide insertions, deletions, and/or substitutions found in other wild-type maize genomes. TALE DNA-binding polypeptides predicted to bind the maize WUS2 promoter target sequences within SEQ ID NO:4 include the polypeptides comprising SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:72, SEQ ID NO:75, and SEQ ID NO:78, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:17, 19, 21, 72, 75, or 78; or one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO: 17, 19, 21, 72, 75, or 78. Artificial transcription factors (ATFs) comprising the aforementioned TALE DNA-binding polypeptides can further comprise operably linked nuclear localization peptides and transcription activation domains. Such ATFs predicted to bind and activate the endogenous ZmWUS2 promoter include the ATFs set forth in SEQ ID NO:18, 20, 22, 73, 76, or 79, as well as variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:18, 20, 22, 73, 76, or 79; or having at one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:18, 20, 22, 73, 76, or 79. Any of the aforementioned ATFs comprising the aforementioned TALE DNA-binding polypeptides predicted to bind the maize WUS2 (ZmWUS2) promoter can be used either independently, in tandem pairs of ATFs predicted to bind at ˜100 bp intervals in the maize WUS2 promoter sequences located 5′ to the ZmWUS2 transcription start site, or as a set of three ATFs predicted to bind at ˜100 bp intervals in ZmWUS2 promoter sequences located 5′ to the ZmWUS2 transcription start site. In other embodiments, ATFs predicted to bind the maize WUS2 promoter can be used in tandem pairs of ATFs predicted to bind at ˜50 bp intervals in ZmWUS2 promoter sequences located 5′ to the ZmWUS2 transcription start site, or as a set of three ATFs predicted to bind at ˜50 bp intervals in ZmWUS2 promoter sequences located 5′ to the ZmWUS2 transcription start site. In other embodiments, target TALE DNA binding sites in the promoter or 5′UTR sequences of other endogenous plant ODP2 or WUS2 genes can be selected and TALE DNA binding domains as well as TALE transcription factors which specifically bind the target binding sites can be designed to obtain TALE transcription factors that can increase expression of other endogenous plant ODP2 or WUS2 genes. In certain embodiments, rules governing the design of TALEs to bind specific DNA sequences that have been published (Moore, Chandrahas, and Bleris 2014; Čermák et al. 2017; Sanjana et al. 2012; Thakore and Gersbach 2016) or provided online (on the https internet site “tale-nt.cac.cornell.edu/node/add/single-tale”) can be applied to the design of the TALE's which bind target TALE binding sites in the ODP2 or WUS2 promoters or 5′UTR.

In certain embodiments, the DNA binding domain can comprise a complex of an RNA guided DNA binding polypeptide that is nuclease activity deficient and a guide RNA comprising a tracrRNA and crRNA polynucleotide sequence which corresponds to a sequence immediately adjacent to the 5′ end of a protospacer adjacent motif (PAM) in the target ODP2 or WUS2 promoter or 5′ UTR, where the complex specifically binds a sequence within the promoter or 5′ untranslated region (5′ UTR) of the endogenous ODP2 or WUS2 gene. RNA guided DNA binding polypeptides that are nuclease activity deficient are also referred to herein as nuclease activity deficient RNA-guided DNA binding polypeptides (ndRGDBP). In certain embodiments, the guide RNA is a single guide RNA (sgRNA) where the crRNA and the tracrRNA are covalently linked. In other embodiments, a dual guide RNA can be used where the crRNA and the tracrRNA are not covalently linked. In general, the crRNA typically comprises about an 18 or 19 to about a 21 or 22 nucleotide sequence which corresponds to the sequence immediately adjacent to the 5′ end of a protospacer adjacent motif (PAM) (e.g., for Cas9 and similar RNA directed nucleases). In general, the crRNA typically comprises about a 20, 21, 22, 23, or 24 nucleotide sequence which corresponds to the sequence immediately adjacent to the 3′ end of a PAM (e.g., for Cas12a (i.e., Cpf1) and similar RNA directed nucleases). Nuclease activity deficient RNA guided DNA binding polypeptides (ndRGDBP) used in such complexes can comprise RNA guided nucleases (Cas or Cas12a nucleases) having mutations that render the protein nuclease activity deficient (e.g., having a 99% or greater reduction in nuclease activity under physiological conditions in a plant cell nucleus). Such nuclease deficient variants of Cas like Cas9 or Cas12a proteins are referred to herein and elsewhere as “dCas” (e.g., dCas9, dCasJ, and the like) or “dCpf1” or “dCas12a” proteins (i.e., “dead Cas” or “dead Cpf1” or “dead Cas12a”). Domains in Cas or Cas12a proteins which can be disrupted to reduce or eliminate nuclease activity include HNH and RuvC-like nuclease domains. Mutations in the catalytic residues of the HNH and RuvC-like nuclease domains of Cas proteins can provide for nuclease-deficient RNA-guided DNA binding proteins (Jinek et al., 2012; Schindele et al., 2018). Examples of such mutations include the D10A and H840A mutations in the Cas9 protein and analogous mutations in the corresponding residues of other Cas9-like proteins identified by alignment with the Cas9 protein. Examples of a dCas9 protein include the polypeptide of SEQ ID NO:29. Other dCas proteins can be obtained by inactivation of nuclease domains include the dCasJ mutants obtained by mutating the CasJ protein of SEQ ID NO:47. Mutations in the nuclease domain of the CasJ include D901A and/or E1228A amino acid substitutions in the CasJ protein of SEQ ID NO:47 and analogous mutations in the corresponding residues of other CasJ proteins identified by alignment with the CasJ protein of SEQ ID NO:47. Mutations in the RuvC-like nuclease domains of Cas12a (i.e., Cpf1) proteins can provide for dCas12a nuclease-deficient RNA-guided DNA binding proteins. Examples of such mutations include the E993A mutation in the AsCpf1 protein (Zhang et al, 2017), the D917A, E1006A, E1028A, D1255A, and/or N1257A mutations in the AsCpf1 protein of SEQ ID NO:44, the D832A, E925A, and/or D1148A mutations in the LbCpf1 protein of SEQ ID NO:45, the D917A, E1006A, E1028A, D1255A, and/or N1257A mutations in the FnCpf1 protein of SEQ ID NO:46, and analogous mutations in the corresponding residues of other Cpf1 proteins identified by alignment with the Cpf1 proteins of SEQ ID NO:44, 45, or 46. Any of the aforementioned dCas9, dCas, dCasJ, or dCpf1 proteins can be used in artificial transcription factors (ATFs) provided herein that further comprise transcription activation domains, cell-penetrating peptides, and nuclear localization domains. Examples of such ATFs include the dCas9 ATF set forth in SEQ ID NO:30, SEQ ID NO:90, and variants thereof that retain RNA guided DNA binding activity and that are nuclease activity deficient. Such dCas9 ATF variants that retain RNA guided DNA binding activity and that are nuclease activity deficient include variants thereof having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the entire length of SEQ ID NO:30 or 90; or having at one or more conservative and/or semi-conservative amino acid substitutions in SEQ ID NO:90. Such artificial transcription factors are used with guide RNAs (sgRNAs or crRNAs and a tracrRNA) to form an ATF/guide RNA complex which can specifically bind sequences in the plant ODP2 and/or WUS2 promoters or 5′ UTR that is immediately adjacent to a protospacer adjacent motif (PAM) sequence. Guide RNAs that direct the dCas or dCpf1 ATF proteins to endogenous plant ODP2 or WUS2 genes can be obtained by identifying target sequences adjacent to PAM sequences in the plant ODP2 and/or WUS2 promoters or 5′ UTR and synthesizing a crRNA or sgRNA that is complementary to that sequence. The type of RNA-guided DNA binding program typically informs the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g., 5′-NGG are typically targeted for design of crRNAs or sgRNAs used with dCas9 proteins. T-rich PAM sites (e.g., 5′-TTTV [1], where “V” is A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with dCpf1 proteins. PAM sites including TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN targeted for design of crRNAs or sgRNAs used with dCasJ proteins (e.g., SEQ ID NO:47). Such crRNAs or sgRNAs can be complementary to sequences that are immediately adjacent to PAM sequences located on either strand of the OPD2 or WUS2 promoter. In certain embodiments the dCas or dCpf1 ATF proteins and guide RNAs are provided to the cell as a pre-assembled ribonucleoprotein (RNP) complex. For example, the ATF can be expressed in an expression host (e.g., E. coli), purified, and complexed with the guide RNA. In other embodiments, the dCas or dCpf1 ATF proteins and guide RNAs are provided separately to the plant cell. Guide RNAs that are synthesized and optionally including chemically modified ribonucleotides can also be used (O'Reilly et al., 2018; Yin et al., 2018). In other embodiments, the dCas or dCpf1 ATF proteins and guide RNAs are provided by introducing one or more polynucleotides encoding the dCas or dCpf1 ATF proteins and/or guide RNA(s) into the target plant cell. In certain embodiments, the guide RNAs are provided to the plant cell by introducing polynucleotides comprising a class III RNA polymerase III promoter that is operably linked to the DNA encoding the guide RNA (Long et al., 2018). Such RNA polymerase III promoters include U6 promoters from monocot plants (e.g., OsU6a, OsU6b, and OsU6c from rice) or dicot plants (e.g., GmU6 from soybean, GhU6 from cotton, and AtU6-1 or AtU6-29 from Arabidopsis thaliana). Useful U6 promoters from maize, tomato, or soybean are disclosed in WO 2015/131101, which is incorporated herein by reference in its entirety with respect to such promoters and their use. In certain embodiments, the plant cell is a maize plant and an ATF/guide RNA complex that specifically binds target DNA sequence located within the promoter or 5′ untranslated region (5′ UTR) of the endogenous maize ODP2 or maize WUS2 gene. Expression of the guide RNA can in certain embodiments be driven by a plant U6 spliceosomal RNA promoter, which can be native to the genome of the plant cell or from a different species, and claiming priority to U.S. Provisional Patent Application 61/945,700, incorporated herein by reference, or a homologue thereof; such a promoter is operably linked to DNA encoding the guide RNA for directing an endonuclease, followed by a suitable 3′ element such as a U6 poly-T terminator. In another embodiment, an expression cassette for expressing guide RNAs in plants is used, wherein the promoter is a plant U3, 7SL (signal recognition particle RNA), U2, or U5 promoter, or chimerics thereof, e.g., as described in WO 2015/131101), incorporated herein by reference. Guide RNAs that can be used to target a dCas9 ATF to an endogenous maize ODP2 promoter can comprise crRNA molecules encoded by DNA molecules set forth as SEQ ID NO:31, 32, 33, 34, or 35. In certain embodiments, the dCas9 ATF and only one guide RNA comprising a crRNA encoded by SEQ ID NO:31, 32, 33, 34, or 35 are introduced into the maize plant cell to activate transcription of the endogenous maize ODP2 gene. In other embodiments, the dCas9 ATF and two, three, four, or five guide RNAs each comprising one crRNA molecules encoded by SEQ ID NO:31, 32, 33, 34, and/or 35 are introduced into the maize plant cell to activate transcription of the endogenous maize ODP2 gene. Guide RNAs that can be used to target a dCas9 ATF to an endogenous maize WUS2 promoter can comprise crRNA molecules encoded by DNA molecules set forth as SEQ ID NO:36, 37, 38, 39, or 40. In certain embodiments, the dCas9 ATF and only one guide RNA comprising a crRNA encoded by SEQ ID NO: 36, 37, 38, 39, or 40 are introduced into the maize plant cell to activate transcription of the endogenous maize WUS2 gene. In other embodiments, the dCas9 ATF and two, three, four, or five guide RNAs each comprising one crRNA molecule encoded by SEQ ID NO:31, 32, 33, 34, and/or 35 are introduced into the maize plant cell to activate transcription of the endogenous maize WUS2 gene. In certain embodiments, the crRNAs SEQ ID NO:31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 further comprise a covalently linked tracrRNA and are thus provided as sgRNAs. In other embodiments, the crRNAs SEQ ID NO:31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 are provided with a non-covalently linked tracrRNA to provide a dual guide RNA. In instances where an allelic variant of an endogenous maize ODP2 or WUS2 promoter that differs in sequence by one or more insertions, deletions, and/or substitutions from SEQ ID NO:3 or SEQ ID NO:4, respectively, a corresponding crRNA can be synthesized that is complementary to the allelic variant sequence and used in a single or dual guide RNA with a dCas ATF to activate transcription of the endogenous maize ODP2 gene or WUS2 gene that comprises the allelic variant promoter sequence.

In certain embodiments, the expression of the endogenous ODP2 and/or WUS2 genes are increased in isolated plant cells or plant protoplasts (i.e., are not located in undissociated or intact plant tissues, plant parts, or whole plants). In certain embodiments, the plant cells are obtained from any plant part or tissue or callus. In certain embodiments, the culture includes plant cells obtained from a plant tissue, a cultured plant tissue explant, whole plant, intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, seedling, whole seed, halved seed or other seed fragment, zygotic embryo, somatic embryo, immature embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, callus, or plant cell suspension. In certain embodiments, the plant cell is derived from the L1 or L2 layer of an immature or mature embryo of a monocot plant (e.g., maize, wheat, sorghum, or rice).

In certain embodiments, the expression of the endogenous ODP2 and/or WUS2 genes are increased in plant cells that are located in undissociated or intact plant tissues, plant parts, plant explants, or whole plants. In certain embodiments, the plant cell can be located in an intact nodal bud, a cultured plant tissue explant, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, seedling, whole seed, halved seed or other seed fragment, zygotic embryo, somatic embryo, immature embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, or callus. In certain embodiments, the explants used include immature embryos. Immature embryos (e.g., immature maize embryos) include, 1.8-2.2 mm embryos, 1-7 mm embryos, and 3-7 mm embryos. In certain embodiments, the aforementioned embryos are obtained from mature ear-derived seed, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, tassels, immature ears, and silks. In various aspects, the plant-derived explant used for transformation includes immature embryos, 1.8-2.2 mm embryos, 1-7 mm embryos, and 3.5-7 mm embryos. In an aspect, the embryos used in the disclosed methods can be derived from mature ear-derived seed, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, tassel, immature ear, or silks. In certain embodiments, the plant cell is a pluripotent plant cell (e.g., a stem cell or meristem cell). In certain embodiments, the plant cell is located within the L1 or L2 layer of an immature or mature embryo of a monocot plant (e.g., maize, wheat, sorghum, or rice). In certain embodiments, methods of editing genomes of whole plants, seeds, embryos, explants, or meristematic tissue published in WO2018085693, which is incorporated herein by reference in its entirety, can be adapted for use in the plant cells and related systems, methods, compositions, or cultures provided herein.

In certain embodiments, the plant cells can comprise haploid, diploid, or polyploid plant cells or plant protoplasts, for example, those obtained from a haploid, diploid, or polyploid plant, plant part or tissue, or callus. In certain embodiments, plant cells in culture (or the regenerated plant, progeny seed, and progeny plant) are haploid or can be induced to become haploid; techniques for making and using haploid plants and plant cells are known in the art, see, e.g., methods for generating haploids in Arabidopsis thaliana by crossing of a wild-type strain to a haploid-inducing strain that expresses altered forms of the centromere-specific histone CENH3, as described by Maruthachalam and Chan in “How to make haploid Arabidopsis thaliana”, protocol available at www[dot]openwetware[dot]org/images/d/d3/Haploid_Arabidopsis_protocol[dot]pdf; (Ravi et al. (2014) Nature Communications, 5:5334, doi: 10.1038/ncomms6334). Haploids can also be obtained in a wide variety of monocot plants (e.g., maize, wheat, rice, sorghum, barley) or dicot plants (e.g., soybean, Brassica sp. including canola, cotton, tomato) by crossing a plant comprising a mutated CENH3 gene with a wildtype diploid plant to generate haploid progeny as disclosed in U.S. Pat. No. 9,215,849, which is incorporated herein by reference in its entirety. Haploid-inducing maize lines that can be used to obtain haploid maize plants and/or cells include Stock 6, MHI (Moldovian Haploid Inducer), indeterminate gametophyte (ig) mutation, KEMS, RWK, ZEM, ZMS, KMS, and well as transgenic haploid inducer lines disclosed in U.S. Pat. No. 9,677,082, which is incorporated herein by reference in its entirety. Examples of haploid cells include but are not limited to plant cells obtained from haploid plants and plant cells obtained from reproductive tissues, e.g., from flowers, developing flowers or flower buds, ovaries, ovules, megaspores, anthers, pollen, megagametophyte, and microspores. In certain embodiments where the plant cell or plant protoplast is haploid, the genetic complement can be doubled by chromosome doubling (e.g., by spontaneous chromosomal doubling by meiotic non-reduction, or by using a chromosome doubling agent such as colchicine, oryzalin, trifluralin, pronamide, nitrous oxide gas, anti-microtubule herbicides, anti-microtubule agents, and mitotic inhibitors) in the plant cell or plant protoplast to produce a doubled haploid plant cell or plant protoplast wherein the complement of genes or alleles is homozygous; yet other embodiments include regeneration of a doubled haploid plant from the doubled haploid plant cell or plant protoplast. Another embodiment is related to a hybrid plant having at least one parent plant that is a doubled haploid plant provided by this approach. Production of doubled haploid plants provides homozygosity in one generation, instead of requiring several generations of self-crossing to obtain homozygous plants. The use of doubled haploids is advantageous in any situation where there is a desire to establish genetic purity (i.e. homozygosity) in the least possible time. Doubled haploid production can be particularly advantageous in slow-growing plants, such as fruit and other trees, or for producing hybrid plants that are offspring of at least one doubled-haploid plant.

In certain embodiments, the plant cells where expression of the endogenous ODP2 and/or WUS2 genes are increased, as well as the related methods, systems, compositions, or reaction mixtures provided herein can include plant cells obtained from or located in any monocot or dicot plant species of interest, for example, row crop plants, fruit-producing plants and trees, vegetables, trees, and ornamental plants including ornamental flowers, shrubs, trees, groundcovers, and turf grasses. In certain non-limiting embodiments, the plant cells are obtained from or located in alfalfa (Medicago sativa), almonds (Prunus dulcis), apples (Malus×domestica), apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica), asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cacao (Theobroma cacao), canola and rapeseed or oilseed rape, (Brassica napus), carnation (Dianthus caryophyllus), carrots (Daucus carota sativus), cassava (Manihot esculentum), cherry (Prunus avium), chickpea (Cider arietinum), chicory (Cichorium intybus), chili peppers and other capsicum peppers (Capsicum annuum, C. frutescens, C. chinense, C. pubescens, C. baccatum), chrysanthemums (Chrysanthemum spp.), coconut (Cocos nucifera), coffee (Coffea spp. including Coffea arabica and Coffea canephora), cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata), cucumber (Cucumis sativus), currants and gooseberries (Ribes spp.), eggplant or aubergine (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linum usitatissumum L.), geraniums (Pelargonium spp.), grapefruit (Citrus×paradisi), grapes (Vitus spp.) including wine grapes (Vitus vinifera), guava (Psidium guajava), hemp and cannabis (e.g., Cannabis sativa and Cannabis spp.), hops (Humulus lupulus), irises (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa), limes (Citrus spp.), maize (Zea mays L.), mango (Mangifera indica), mangosteen (Garcinia mangostana), melon (Cucumis melo), millets (Setaria spp, Echinochloa spp, Eleusine spp, Panicum spp., Pennisetum spp.), oats (Avena sativa), oil palm (Ellis quineensis), olive (Olea europaea), onion (Allium cepa), orange (Citrus sinensis), papaya (Carica papaya), peaches and nectarines (Prunus persica), pear (Pyrus spp.), pea (Pisa sativum), peanut (Arachis hypogaea), peonies (Paeonia spp.), petunias (Petunia spp.), pineapple (Ananas comosus), plantains (Musa spp.), plum (Prunus domestica), poinsettia (Euphorbia pulcherrima), Polish canola (Brassica rapa), poplar (Populus spp.), potato (Solanum tuberosum), pumpkin (Cucurbita pepo), rice (Oryza sativa L.), roses (Rosa spp.), rubber (Hevea brasiliensis), rye (Secale cereale), safflower (Carthamus tinctorius L), sesame seed (Sesame indium), sorghum (Sorghum bicolor), soybean (Glycine max L.), squash (Cucurbita pepo), strawberries (Fragaria spp., Fragaria×ananassa), sugar beet (Beta vulgaris), sugarcanes (Saccharum spp.), sunflower (Helianthus annus), sweet potato (Ipomoea batatas), tangerine (Citrus tangerina), tea (Camellia sinensis), tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum), tulips (Tulipa spp.), turnip (Brassica rapa rapa), walnuts (Juglans spp. L.), watermelon (Citrulus lanatus), wheat (Tritium aestivum), or yams (Discorea spp.).

In certain embodiments, the plant cells where the expression of the endogenous ODP2 and/or WUS2 genes are increased can be plant cells that are (a) encapsulated or enclosed in or attached to a polymer (e.g., pectin, agarose, or other polysaccharide) or other support (solid or semi-solid surfaces or matrices, or particles or nanoparticles); (b) encapsulated or enclosed in or attached to a vesicle or liposome or other fluid compartment; or (c) not encapsulated or enclosed or attached. In certain embodiments, the plant cells can be in liquid or suspension culture, or cultured in or on semi-solid or solid media, or in a combination of liquid and solid or semi-solid media (e.g., plant cells or protoplasts cultured on solid medium with a liquid medium overlay, or plant cells or protoplasts attached to solid beads or a matrix and grown with a liquid medium). In certain embodiments, the plant cells encapsulated in a polymer (e.g., pectin, agarose, or other polysaccharide) or other encapsulating material, enclosed in a vesicle or liposome, suspended in a mixed-phase medium (such as an emulsion or reverse emulsion), or embedded in or attached to a matrix or other solid support (e.g., beads or microbeads, membranes, or solid surfaces).

In a related aspect, the disclosure provides arrangements of plant cells having improved plant cell regenerative potential in the systems, methods, and compositions described herein, such as arrangements of plant cells convenient for screening purposes or for high-throughput and/or multiplex transformation or gene editing experiments. In an embodiment, the disclosure provides an arrangement of multiple plant cells comprising: (a) an exogenous gene transcription agent which transiently increases expression of an endogenous ODP2 polypeptide and/or an exogenous gene transcription agent which increases expression of an endogenous WUS2 polypeptide; and optionally (b) genome altering reagent(s). In certain embodiments, the arrangements of plant cells can further comprise at least one chemical, enzymatic, or physical delivery agent. In another embodiment, the disclosure provides an array including a plurality of containers, each including at least one plant cell or plant protoplast having improved plant cell regenerative potential. In an embodiment, the disclosure provides arrangements of plant cells having the exogenous gene transcription agent(s) and optionally the genome altering reagents, wherein the plant cells are in an arrayed format, for example, in multi-well plates, encapsulated or enclosed in vesicles, liposomes, or droplets (useful, (e.g., in a microfluidics device), or attached discretely to a matrix or to discrete particles or beads; a specific embodiment is such an arrangement of multiple plant cells having improved plant cell regenerative potential provided in an arrayed format, further including at least one genome altering reagent(s) (e.g., an RNA-guided DNA nuclease, at least one guide RNA, or a ribonucleoprotein including both an RNA-guided DNA nuclease and at least one guide RNA), which may be different for at least some locations on the array or even for each location on the array, and optionally at least one chemical, enzymatic, or physical delivery agent.

In the systems and methods provided herein, plant cells can be exposed to one exogenous gene transcription agent which transiently increases expression of an endogenous ODP2 polypeptide and/or at least one exogenous gene transcription agent which increases expression of an endogenous WUS2 polypeptide and/or genome altering reagents in any temporal order. In certain embodiments, the genome altering reagents and aforementioned exogenous gene transcription agent(s) are provided simultaneously. In other embodiments, the genome altering reagents are provided after the exogenous gene transcription agent(s) are provided. In other embodiments, the genome altering reagents are provided before the exogenous gene transcription agent(s) are provided. In summary, the genome altering reagents can be provided to a plant cell either previous to, concurrently with, or subsequent to exposing the plant cell to the exogenous gene transcription agent(s).

Plant cells having improved plant cell regenerative potential conferred by an increase in the transient expression of the endogenous ODP2 and/or WUS2 genes are provided herein. Also provided by the disclosure are compositions derived from or grown from the plant cell or plant protoplast having improved plant cell regenerative potential, provided by the systems and methods disclosed herein; such compositions include multiple protoplasts or cells, callus, a somatic embryo, a somatic meristem, embryogenic callus, or a regenerated plant grown from the plant cell or plant protoplast having improved plant cell regenerative potential. Improved plant cell regenerative potential in plant cells that have been subjected to a transient increase in ODP2 and/or WUS2 gene expression can be assessed by a variety of techniques. In certain embodiments, such techniques can compare the numbers and/or amount of regenerable plant structures (e.g., immature embryos, somatic embryos, embryogenic calli, somatic meristems, organogenic calli, shoots, or shoots further comprising roots) formed and/or recovered from a given number of plant cells subjected to the transient increase in endogenous ODP2 and/or WUS2 gene expression versus control plant cells that were not subjected to the transient increase in ODP2 and/or WUS2 gene expression. In certain embodiments, it is understood that the plant cells can be directly subjected to the transient increase in endogenous ODP2 and/or WUS2 gene expression (e.g., by or indirectly (e.g., by exposure, contact, or other signaling of neighboring cells The principle attributes of tissues targeted for transient expression of the ATFs provided would be the presence of dividing cells and the ability to grow in tissue culture media. These tissues include, but are not limited to dividing cells from young maize leaf, meristems and scutellar tissue from about 8 or 10 to about 12 or 14 days after pollination (DAP) embryos. The isolation of maize embryos has been described in several publications (Brettschneider, Becker, and Lörz 1997; Leduc et al. 1996; Frame et al. 2011; K. Wang and Frame 2009). In certain embodiments, basal leaf tissues (e.g., leaf tissues located about 0 to 3 cm from the ligule of a maize plant; Kirienko, Luo, and Sylvester 2012) are targeted for transient expression of the ATFs. In certain embodiments, such increases in numbers and/or amounts of regenerable plant structures can be observed in about 1, 2, or 3 to about 7, 10, 14, 30, or 60 days following the transient increase in endogenous ODP2 and/or WUS2 gene expression. Methods for obtaining regenerable plant structures and regenerating plants from the plant cells provided herein can be adapted from methods disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. In certain embodiments, single plant cells subjected to the transient increase in endogenous ODP2 and/or WUS2 gene expression will give rise to single regenerable plant structures. In certain embodiments, the single regenerable plant cell structure can form from a single cell on, or within, an explant that has been subjected to the transient increase in endogenous ODP2 and/or WUS2 gene expression and optionally subjected to treatment with a genome altering reagent. In certain embodiments, initiation or formation of the single plant cell regenerable structure can occur where single-cell-derived cell or tissue proliferation (e.g., growth of callus, non-differentiated callus, embryogenic callus and organogenic callus) occurring before initiation of the regenerable plant structure is reduced or absent. In certain embodiments, regenerable plant structures from plant cells subjected to the transient increase in endogenous ODP2 and/or WUS2 gene expression and optionally a genome altering reagent can be form the regenerable plant structure in the absence of exogenous cytokinin or with levels of cytokinin that are lower than those required to initiate formation of the regenerable structure from a control cell. In certain embodiments, regenerable plant structures from plant cells subjected to the transient increase in endogenous ODP2 and/or WUS2 gene expression and optionally a genome altering reagent can be identified and/or selected via a positive growth selection based on the ability of those plant cells to initiate and/or form the regenerable plant structures more rapidly than adjacent plant cells that have not been subjected to the transient increase in endogenous ODP2 and/or WUS2 gene expression. In certain embodiments, such positive growth selection can obviate or reduce the need to use a traditional negative selection system where an antibiotic or herbicide is used to inhibit growth of adjacent, non-transformed cells that do not contain a gene that confers resistance to the antibiotic or herbicide. Nonetheless, embodiments where a selectable marker gene conferring resistance to an antibiotic, herbicide, or other agent can be introduced into the plant cell at least temporarily during initiation and/or formation of the regenerable plant cell structures to facilitate identification and recovery.

In some embodiments, methods provided herein can include the additional step of growing or regenerating a plant from a plant cell that had been subjected to an increase in the transient expression of the endogenous ODP2 and/or WUS2 genes or from a regenerable plant structure obtained from that plant cell. In certain embodiments, the plant can further comprise an inserted transgene, a target gene edit, or genome edit as provided by the methods and compositions disclosed herein. In certain embodiments, callus is produced from the plant cell, and plantlets and plants produced from such callus. In other embodiments, whole seedlings or plants are grown directly from the plant cell without a callus stage. Thus, additional related aspects are directed to whole seedlings and plants grown or regenerated from the plant cell or plant protoplast having a target gene edit or genome edit, as well as the seeds of such plants. In certain embodiments wherein the plant cell or plant protoplast is subjected to genetic or epigenetic modification (for example, stable or transient expression of a transgene, gene silencing, epigenetic silencing, or genome editing by means of, e.g., an RNA-guided DNA nuclease), the grown or regenerated plant exhibits a phenotype associated with the genetic or epigenetic modification. In certain embodiments, the grown or regenerated plant includes in its genome two or more genetic or epigenetic modifications that in combination provide at least one phenotype of interest. In certain embodiments, a heterogeneous population of plant cells having a target gene edit or genome edit, at least some of which include at least one genetic or epigenetic modification, is provided by the method; related aspects include a plant having a phenotype of interest associated with the genetic or epigenetic modification, provided by either regeneration of a plant having the phenotype of interest from a plant cell or plant protoplast selected from the heterogeneous population of plant cells having a target gene or genome edit, or by selection of a plant having the phenotype of interest from a heterogeneous population of plants grown or regenerated from the population of plant cells having a target gene edit or genome edit. Examples of phenotypes of interest include herbicide resistance, improved tolerance of abiotic stress (e.g., tolerance of temperature extremes, drought, or salt) or biotic stress (e.g., resistance to nematode, bacterial, or fungal pathogens), improved utilization of nutrients or water, modified lipid, carbohydrate, or protein composition, improved flavor or appearance, improved storage characteristics (e.g., resistance to bruising, browning, or softening), increased yield, altered morphology (e.g., floral architecture or color, plant height, branching, root structure). In an embodiment, a heterogeneous population of plant cells having a target gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) is exposed to conditions permitting expression of the phenotype of interest; e.g., selection for herbicide resistance can include exposing the population of plant cells having a target gene edit or genome edit (or seedlings or plants grown or regenerated therefrom) to an amount of herbicide or other substance that inhibits growth or is toxic, allowing identification and selection of those resistant plant cells (or seedlings or plants) that survive treatment. Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can be adapted from published procedures (Roest and Gilissen, Acta Bot. Neerl., 1989, 38(1), 1-23; Bhaskaran and Smith, Crop Sci. 30(6):1328-1337; Ikeuchi et al., Development, 2016, 143: 1442-1451). Methods for obtaining regenerable plant structures and regenerating plants from plant cells or regenerable plant structures can also be adapted from US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. Also provided are heterogeneous populations, arrays, or libraries of such plants, succeeding generations or seeds of such plants grown or regenerated from the plant cells or plant protoplasts, having a target gene edit or genome edit, parts of the plants (including plant parts used in grafting as scions or rootstocks), or products (e.g., fruits or other edible plant parts, cleaned grains or seeds, edible oils, flours or starches, proteins, and other processed products) made from the plants or their seeds. Embodiments include plants grown or regenerated from the plant cells having a target gene edit or genome edit, wherein the plants contain cells or tissues that do not have a genetic or epigenetic modification, e.g., grafted plants in which the scion or rootstock contains a genetic or epigenetic modification, or chimeric plants in which some but not all cells or tissues contain a genetic or epigenetic modification. Plants in which grafting is commonly useful include many fruit trees and plants such as many citrus trees, apples, stone fruit (e.g., peaches, apricots, cherries, and plums), avocados, tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well as various ornamental plants such as roses. Grafted plants can be grafts between the same or different (generally related) species. Additional related aspects include a hybrid plant provided by crossing a first plant grown or regenerated from a plant cell or plant protoplast having a target gene edit or genome edit and having at least one genetic or epigenetic modification, with a second plant, wherein the hybrid plant contains the genetic or epigenetic modification; also contemplated is seed produced by the hybrid plant. Also envisioned as related aspects are progeny seed and progeny plants, including hybrid seed and hybrid plants, having the regenerated plant as a parent or ancestor. The plant cells and derivative plants and seeds disclosed herein can be used for various purposes useful to the consumer or grower. The intact plant itself may be desirable, e.g., plants grown as cover crops or as ornamentals. In other embodiments, processed products are made from the plant or its seeds, such as extracted proteins, oils, sugars, and starches, fermentation products, animal feed or human food, wood and wood products, pharmaceuticals, and various industrial products.

An exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene can be provided to a cell (e.g., a plant cell or plant protoplast) by any suitable technique. In certain embodiments, the exogenous gene transcription agent is provided by directly contacting a plant cell with the exogenous gene transcription agent or the polynucleotide that encodes the exogenous gene transcription agent. In certain embodiments, the exogenous gene transcription agent is provided by transporting the exogenous gene transcription agent or a polynucleotide that encodes exogenous gene transcription agent into a plant cell or plant protoplast using a chemical, enzymatic, or physical agent. In certain embodiments, the exogenous gene transcription agent is provided by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of a plant cell or plant protoplast with a polynucleotide encoding the exogenous gene transcription agent; see, e.g., Broothaerts et al. (2005) Nature, 433:629-633. In an embodiment, the exogenous gene transcription agent is provided by transcription in a plant cell or plant protoplast of a DNA that encodes the exogenous gene transcription agent and is stably integrated in the genome of the plant cell or is provided to the plant cell or plant protoplast in the form of a plasmid or expression vector (e.g., a viral vector) that encodes the exogenous gene transcription agent. In certain embodiments, the exogenous gene transcription agent is provided to the plant cell or plant protoplast as a polynucleotide that encodes exogenous gene transcription agent, e.g., in the form of an RNA (e.g., mRNA or RNA containing an internal ribosome entry site (IRES)) encoding the exogenous gene transcription agent. Genome altering reagents can also be introduced into the plant cells by similar techniques.

Transient expression of an exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene (e.g., expression of an guide RNA from a DNA, or expression and translation of an ATF or RNA-guided DNA binding polypeptide from a DNA encoding the ATF or polypeptide), can be achieved by a variety of techniques. Certain embodiments are useful in effectuating transient expression of the endogenous ODP2 and/or WUS2 gene without remnants of the exogenous gene transcription agents that provide for the transient expression or selective genetic markers occurring in progeny. In certain embodiments, the exogenous gene transcription agents are provided directly to the plant cells, systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of a cell free synthetic process (e.g., in vitro translation), or as isolated or semi-purified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, artificial transcription factors (ATFs) are targeted to the plant cell or cell nucleus in a manner that insures transient expression (e.g., by methods adapted from Gao et al. 2016; or Li et al. 2009). In certain embodiments, the exogenous gene transcription agent is delivered into the plant cell by delivery of the agent itself in the absence of any polynucleotide that encodes the agent. Examples of exogenous gene transcription agents that can be delivered in the absence of any encoding polynucleotides include polypeptide ATFs (e.g., aZFPs or aTALEs), RNA-guided DNA binding polypeptide, and RNA guides. RNA-guided DNA binding polypeptide/RNA guides can be delivered separately and/or as RNP complexes. In certain embodiments, ATF proteins can be produced in a heterologous system, purified and delivered to plant cells by particle bombardment (e.g., by methods adapted from Martin-Ortigosa and Wang 2014). In embodiments where the exogenous gene transcription agents are delivered in the absence of any encoding polynucleotides, the delivered agent is expected to degrade over time in the absence of ongoing expression from any introduced encoding polynucleotides to result in transient endogenous ODP2 gene and/or the endogenous WUS2 gene expression. In certain embodiments, the exogenous gene transcription agent is delivered into the plant cell by delivery of a polynucleotide that encodes the agent. In certain embodiments, ATFs can be encoded on a bacterial plasmid and delivered to plant tissue by particle bombardment (e.g., by methods adapted from Hamada et al. 2018; or Kirienko, Luo, and Sylvester 2012). In certain embodiments, ATFs can be encoded on a T-DNA and transiently transferred to plant cells using agrobacterium (e.g., by methods adapted from Leonelli et al. 2016; or Wu et al. 2014). In certain embodiments, ATFs can be encoded in a viral genome and delivered to plants (e.g., by methods adapted from Honig et al. 2015). In certain embodiments, ATFs can be encoded in mRNA or an RNA comprising an IRES and delivered to target plant cells. In certain embodiments where the exogenous gene transcription agent comprises an RNA-guided DNA binding polypeptide and an RNA guide, the polypeptide or guide can be delivered by a combination of: (i) an encoding polynucleotide for either polypeptide or the guide; and (ii) either polypeptide or the guide itself in the absence of an encoding polynucleotide. In certain embodiments, the exogenous gene transcription agent is delivered into the plant cell by delivery of a polynucleotide that encodes the agent. In certain embodiments, the polynucleotide that encodes the exogenous gene transcription agent is not integrated into a plant cell genome (e.g., as a polynucleotide lacking sequences that provide for integration, by agroinfiltration on an integration deficient T-DNA vector or system, or in a viral vector), is not operably linked to polynucleotides which provide for autonomous replication, and/or only provided with factors (e.g., viral replication proteins) that provide for autonomous replication. Suitable techniques for transient expression including biolistic and other delivery of polynucleotides, agroinfiltration, and use of viral vectors disclosed by Canto, 2016 and others can be adapted for transient expression of the agents provided herein. Transient expression of the agent encoded by a non-integrated polynucleotide effectuated by excision of the polynucleotide and/or regulated expression of the agent. In certain embodiments, the polynucleotide that encodes the exogenous gene transcription agent is integrated into a plant cell genome (e.g., a nuclear or plastid genome) and transient expression of the agent is effectuated by excision of the polynucleotide and/or regulated expression of the agent. Excision of a polynucleotide encoding the agent can be provided by use of site-specific recombination systems (e.g., Cre-Lox, FLP-FRT). Regulated expression of the agent can be effectuated by methods including: (i) operable linkage of the polynucleotide encoding the agent to a developmentally-regulated, de-repressable, and/or inducible promoter; and/or (ii) introduction of a polynucleotide (e.g., dsRNA or amiRNA) that can induce siRNA-mediated inhibition of the agent. Suitable site-specific recombination systems as well as developmentally-regulated, de-repressable, and/or inducible promoters include those disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. In any of the aforementioned embodiments, transient expression of the endogenous ODP2 and/or WUS2 genes can also be achieved by using an exogenous gene transcription agent comprising a DNA binding domain or complex and a transcription activation domain (TAD) that can be operably associated through binding a common ligand (e.g., the iDimerize™ Regulated Transcription System that uses Dmr A, B, or C dimerization domains and ligands; Takara Bio, USA, Inc.). In such embodiments, transient expression of the endogenous ODP2 and/or WUS2 genes can occur upon addition of the common ligand.

Polynucleotides that can be used to effectuate transient expression of an exogenous gene transcription agent (e.g., a polynucleotide encoding an ATF, RNA-guided DNA binding polypeptide, and/or a guide RNA) include: (a) double-stranded RNA; (b) single-stranded RNA; (c) chemically modified RNA; (d) double-stranded DNA; (e) single-stranded DNA; (f) chemically modified DNA; or (g) a combination of (a)-(f). Certain embodiments of the polynucleotide further include additional nucleotide sequences that provide useful functionality; non-limiting examples of such additional nucleotide sequences include an aptamer or riboswitch sequence, nucleotide sequence that provides secondary structure such as stem-loops or that provides a sequence-specific site for an enzyme (e.g., a sequence-specific recombinase or endonuclease site), T-DNA (e.g., DNA sequence encoding an exogenous gene transcription agent is enclosed between left and right T-DNA borders from Agrobacterium spp. or from other bacteria that infect or induce tumors in plants), a DNA nuclear-targeting sequence, a regulatory sequence such as a promoter sequence, and a transcript-stabilizing or -destabilizing sequence. Certain embodiments of the polynucleotide include those wherein the polynucleotide is complexed with, or covalently or non-covalently bound to, a non-nucleic acid element, e.g., a carrier molecule, an antibody, an antigen, a viral movement protein, a cell-penetrating or pore-forming peptide, a polymer, a detectable label, a quantum dot, or a particulate or nanoparticulate.

Various treatments are useful in delivery of an exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene to a plant cell. In certain embodiments, one or more treatments is employed to deliver the agent (e.g., comprising a polynucleotide, polypeptide or combination thereof) into a plant cell or plant protoplast, e.g., through barriers such as a cell wall, a plasma membrane, a nuclear envelope, and/or other lipid bilayer. In certain embodiments, a polynucleotide-, polypeptide-, or RNP-containing composition comprising the agent(s) are delivered directly, for example by direct contact of the composition with a plant cell. Aforementioned compositions can be provided in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant, plant part, plant cell, or plant explant (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a plant cell or plant protoplast is soaked in a liquid exogenous gene transcription agent-containing composition, whereby the agent is delivered to the plant cell. In certain embodiments, the agent-containing composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In certain embodiments, the agent-containing composition is introduced into a plant cell or plant protoplast, e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in US Published Patent Application 2014/0287509, incorporated by reference in its entirety herein. Other techniques useful for delivering the agent-containing composition to a plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In certain embodiments, the agent-containing composition is provided by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the agent (e.g., ATF, RNA guided ATF, and/or guide RNA); see, e.g., Broothaerts et al. (2005) Nature, 433:629-633. Any of these techniques or a combination thereof are alternatively employed on the plant explant, plant part or tissue or intact plant (or seed) from which a plant cell is optionally subsequently obtained or isolated; in certain embodiments, the agent-containing composition is delivered in a separate step after the plant cell has been isolated. In certain embodiments, the aforementioned methods can also be used to introduce a genome altering reagent into the plant cell.

In embodiments, a treatment employed in delivery of a exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene to a plant cell is carried out under a specific thermal regime, which can involve one or more appropriate temperatures, e.g., chilling or cold stress (exposure to temperatures below that at which normal plant growth occurs), or heating or heat stress (exposure to temperatures above that at which normal plant growth occurs), or treating at a combination of different temperatures. In certain embodiments, a specific thermal regime is carried out on the plant cell, or on a plant, plant explant, or plant part from which a plant cell or plant protoplast is subsequently obtained or isolated, in one or more steps separate from the agent delivery. In certain embodiments, the aforementioned methods can also be used to introduce a genome altering reagent into the plant cell.

In certain embodiments of the plant parts, systems, methods, and compositions provided herein, a whole plant or plant part or seed, or an isolated plant cell, a plant explant, or the plant or plant part from which a plant cell or plant protoplast is obtained or isolated, is treated with one or more delivery agents which can include at least one chemical, enzymatic, or physical agent, or a combination thereof. In certain embodiments, an exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene further includes one or more than one chemical, enzymatic, or physical agents for delivery. Treatment with the chemical, enzymatic or physical agent can be carried out simultaneously with the agent delivery or in one or more separate steps that precede or follow the agent delivery. In certain embodiments, a chemical, enzymatic, or physical agent, or a combination of these, is associated or complexed with the polynucleotide composition, with the donor template polynucleotide, with the exogenous gene transcription agent; examples of such associations or complexes include those involving non-covalent interactions (e.g., ionic or electrostatic interactions, hydrophobic or hydrophilic interactions, formation of liposomes, micelles, or other heterogeneous composition) and covalent interactions (e.g., peptide bonds, bonds formed using cross-linking agents). In non-limiting examples, the exogenous gene transcription agent is provided as a liposomal complex with a cationic lipid; the exogenous gene transcription agent is provided as a complex with a carbon nanotube; and/or exogenous gene transcription agent is provided as a fusion protein between the agent and a cell-penetrating peptide. Examples of agents useful for delivering the exogenous gene transcription agent(s) include the various cationic liposomes and polymer nanoparticles reviewed by Zhang et al. (2007) J. Controlled Release, 123:1-10, and the cross-linked multilamellar liposomes described in US Patent Application Publication 2014/0356414 A1, incorporated by reference in its entirety herein. In any of the aforementioned embodiments, it is further contemplated that the aforementioned methods can also be used to introduce a genome altering reagent into the plant cell.

In certain embodiments, the chemical agent used to deliver an exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene can comprise:

-   -   (a) solvents (e.g., water, dimethylsulfoxide, dimethylformamide,         acetonitrile, N-pyrrolidine, pyridine, hexamethylphosphoramide,         alcohols, alkanes, alkenes, dioxanes, polyethylene glycol, and         other solvents miscible or emulsifiable with water or that will         dissolve phosphonucleotides in non-aqueous systems);     -   (b) fluorocarbons (e.g., perfluorodecalin,         perfluoromethyldecalin);     -   (c) glycols or polyols (e.g., propylene glycol, polyethylene         glycol);     -   (d) surfactants, including cationic surfactants, anionic         surfactants, non-ionic surfactants, and amphiphilic surfactants,         e.g., alkyl or aryl sulfates, phosphates, sulfonates, or         carboxylates; primary, secondary, or tertiary amines; quaternary         ammonium salts; sultaines, betaines; cationic lipids;         phospholipids; tallowamine; bile acids such as cholic acid; long         chain alcohols; organosilicone surfactants including nonionic         organosilicone surfactants such as trisiloxane ethoxylate         surfactants or a silicone polyether copolymer such as a         copolymer of polyalkylene oxide modified heptamethyl trisiloxane         and allyloxypolypropylene glycol methylether (commercially         available as SILWET L-77™ brand surfactant having CAS Number         27306-78-1 and EPA Number CAL. REG. NO. 5905-50073-AA, Momentive         Performance Materials, Inc., Albany, N.Y.); specific examples of         useful surfactants include sodium lauryl sulfate, the Tween         series of surfactants, Triton-X100, Triton-X114, CHAPS and         CHAPSO, Tergitol-type NP-40, Nonidet P-40;     -   (e) lipids, lipoproteins, lipopolysaccharides;     -   (f) acids, bases, caustic agents;     -   (g) peptides, proteins, or enzymes (e.g., cellulase, pectolyase,         maceroenzyme, pectinase), including cell-penetrating or         pore-forming peptides (e.g., (BO100)2K8, Genscript; poly-lysine,         poly-arginine, or poly-homoarginine peptides; gamma zein, see US         Patent Application publication 2011/0247100, incorporated herein         by reference in its entirety; transcription activator of human         immunodeficiency virus type 1 (“HIV-1 Tat”) and other Tat         proteins, see, e.g.,         www[dot]lifetein[dot]com/Cell_Penetrating_Peptides[dot]html and         Jarver (2012) Mol. Therapy—Nucleic Acids, 1:e27, 1-17);         octa-arginine or nona-arginine; poly-homoarginine (see Unnamalai         et al. (2004) FEBS Letters, 566:307-310); see also the database         of cell-penetrating peptides CPPsite 2.0 publicly available at         crdd[dot]osdd[dot]net/raghava/cppsite/     -   (h) RNase inhibitors;     -   (i) cationic branched or linear polymers such as chitosan,         poly-lysine, DEAE-dextran, polyvinylpyrrolidone (“PVP”), or         polyethylenimine (“PEI”, e.g., PEI, branched, MW 25,000, CAS         #9002-98-6; PEI, linear, MW 5000, CAS #9002-98-6; PEI linear, MW         2500, CAS #9002-98-6);     -   (j) dendrimers (see, e.g., US Patent Application Publication         2011/0093982, incorporated herein by reference in its entirety);     -   (k) counter-ions, amines or polyamines (e.g., spermine,         spermidine, putrescine), osmolytes, buffers, and salts (e.g.,         calcium phosphate, ammonium phosphate);     -   (l) polynucleotides (e.g., non-specific double-stranded DNA,         salmon sperm DNA);     -   (m) transfection agents (e.g., Lipofectin®, Lipofectamine®, and         Oligofectamine®, and Invivofectamine® (all from Thermo Fisher         Scientific, Waltham, MA), PepFect (see Ezzat et al. (2011)         Nucleic Acids Res., 39:5284-5298), Transit® transfection         reagents (Minis Bio, LLC, Madison, WI), and poly-lysine,         poly-homoarginine, and poly-arginine molecules including         octo-arginine and nono-arginine as described in Lu et         al. (2010) J. Agric. Food Chem., 58:2288-2294);     -   (n) antibiotics, including non-specific DNA         double-strand-break-inducing agents (e.g., phleomycin,         bleomycin, talisomycin); and/or     -   (o) antioxidants (e.g., glutathione, dithiothreitol, ascorbate).

In any of the aforementioned embodiments, it is further contemplated that the aforementioned chemical agents can also be used to introduce a genome altering reagent into the plant cell.

In certain embodiments, the chemical agent is provided simultaneously with the exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene. In certain embodiments, exogenous gene transcription agent is covalently or non-covalently linked or complexed with one or more chemical agents; for example, an ATF or RNA guided DNA binding protein can be covalently linked to a peptide or protein (e.g., a cell-penetrating peptide or a pore-forming peptide) or non-covalently complexed with cationic lipids, polycations (e.g., polyamines), or cationic polymers (e.g., PEI). In certain embodiments, the exogenous gene transcription agent is complexed with one or more chemical agents to form, e.g., a solution, liposome, micelle, emulsion, reverse emulsion, suspension, colloid, or gel. In any of the aforementioned embodiments, it is further contemplated that genome altering reagents comprising polynucleotides and/or polypeptides can be also be delivered as described above.

In certain embodiments, the physical agent for delivery of an exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene is at least one selected from the group consisting of particles or nanoparticles (e.g., particles or nanoparticles made of materials such as carbon, silicon, silicon carbide, gold, tungsten, polymers, or ceramics) in various size ranges and shapes, magnetic particles or nanoparticles (e.g., silenceMag Magnetotransfection™ agent, OZ Biosciences, San Diego, CA), abrasive or scarifying agents, needles or microneedles, matrices, and grids. In certain embodiments, particulates and nanoparticulates are useful in delivery of the exogenous gene transcription agent. Useful particulates and nanoparticles include those made of metals (e.g., gold, silver, tungsten, iron, cerium), ceramics (e.g., aluminum oxide, silicon carbide, silicon nitride, tungsten carbide), polymers (e.g., polystyrene, polydiacetylene, and poly(3,4-ethylenedioxythiophene) hydrate), semiconductors (e.g., quantum dots), silicon (e.g., silicon carbide), carbon (e.g., graphite, graphene, graphene oxide, or carbon nanosheets, nanocomplexes, or nanotubes), and composites (e.g., polyvinylcarbazole/graphene, polystyrene/graphene, platinum/graphene, palladium/graphene nanocomposites). In certain embodiments, such particulates and nanoparticulates are further covalently or non-covalently functionalized, or further include modifiers or cross-linked materials such as polymers (e.g., linear or branched polyethylenimine, poly-lysine), polynucleotides (e.g., DNA or RNA), polysaccharides, lipids, polyglycols (e.g., polyethylene glycol, thiolated polyethylene glycol), polypeptides or proteins, and detectable labels (e.g., a fluorophore, an antigen, an antibody, or a quantum dot). In various embodiments, such particulates and nanoparticles are neutral, or carry a positive charge, or carry a negative charge. Embodiments of compositions including particulates include those formulated, e.g., as liquids, colloids, dispersions, suspensions, aerosols, gels, and solids. Embodiments include nanoparticles affixed to a surface or support, e.g., an array of carbon nanotubes vertically aligned on a silicon or copper wafer substrate. Embodiments include polynucleotide compositions including particulates (e.g., gold or tungsten or magnetic particles) delivered by a Biolistic-type technique or with magnetic force. The size of the particles used in Biolistics is generally in the “microparticle” range, for example, gold microcarriers in the 0.6, 1.0, and 1.6 micrometer size ranges (see, e.g., instruction manual for the Helios® Gene Gun System, Bio-Rad, Hercules, CA; Randolph-Anderson et al. (2015) “Sub-micron gold particles are superior to larger particles for efficient Biolistic® transformation of organelles and some cell types”, Bio-Rad US/EG Bulletin 2015), but successful Biolistics delivery using larger (40 nanometer) nanoparticles has been reported in cultured animal cells; see O'Brian and Lummis (2011) BMC Biotechnol., 11:66-71. Other embodiments of useful particulates are nanoparticles, which are generally in the nanometer (nm) size range or less than 1 micrometer, e.g., with a diameter of less than about 1 nm, less than about 3 nm, less than about 5 nm, less than about 10 nm, less than about 20 nm, less than about 40 nm, less than about 60 nm, less than about 80 nm, and less than about 100 nm. Specific, non-limiting embodiments of nanoparticles commercially available (all from Sigma-Aldrich Corp., St. Louis, MO) include gold nanoparticles with diameters of 5, 10, or 15 nm; silver nanoparticles with particle sizes of 10, 20, 40, 60, or 100 nm; palladium “nanopowder” of less than 25 nm particle size; single-, double-, and multi-walled carbon nanotubes, e.g., with diameters of 0.7-1.1, 1.3-2.3, 0.7-0.9, or 0.7-1.3 nm, or with nanotube bundle dimensions of 2-10 nm by 1-5 micrometers, 6-9 nm by 5 micrometers, 7-15 nm by 0.5-10 micrometers, 7-12 nm by 0.5-10 micrometers, 110-170 nm by 5-9 micrometers, 6-13 nm by 2.5-20 micrometers. In certain embodiments, physical agents for delivery of an exogenous gene transcription agents can include materials such as gold, silicon, cerium, or carbon, e.g., gold or gold-coated nanoparticles, silicon carbide whiskers, carborundum, porous silica nanoparticles, gelatin/silica nanoparticles, nanoceria or cerium oxide nanoparticles (CNPs), carbon nanotubes (CNTs) such as single-, double-, or multi-walled carbon nanotubes and their chemically functionalized versions (e.g., carbon nanotubes functionalized with amide, amino, carboxylic acid, sulfonic acid, or polyethylene glycol moeities), and graphene or graphene oxide or graphene complexes. Such physical agents that can be adapted for delivery of exogenous gene transcription agents include those disclosed in Wong et al. (2016) Nano Lett., 16:1161-1172; Giraldo et al. (2014) Nature Materials, 13:400-409; Shen et al. (2012) Theranostics, 2:283-294; Kim et al. (2011) Bioconjugate Chem., 22:2558-2567; Wang et al. (2010) J. Am. Chem. Soc. Comm., 132:9274-9276; Zhao et al. (2016) Nanoscale Res. Lett., 11:195-203; and Choi et al. (2016) J. Controlled Release, 235:222-235. See also, for example, the various types of particles and nanoparticles, their preparation, and methods for their use, e.g., in delivering polynucleotides and polypeptides to cells, disclosed in US Patent Application Publications 2010/0311168, 2012/0023619, 2012/0244569, 2013/0145488, 2013/0185823, 2014/0096284, 2015/0040268, 2015/0047074, and 2015/0208663, all of which are incorporated herein by reference in their entirety. In any of the aforementioned embodiments, it is further contemplated that genome altering reagents comprising polynucleotides and/or polypeptides can be also be delivered as described above.

In certain embodiments wherein the exogenous gene transcription agents comprise a gRNA (or polynucleotide encoding the gRNA) is provided in a composition that further includes an RNA guided DNA binding polypeptide that is nuclease activity deficient (or a polynucleotide that encodes the same), one or more one chemical, enzymatic, or physical agent can similarly be employed. In certain embodiments, the RNA guide and the nuclease activity deficient RNA-guided DNA binding polypeptide (ndRGDBP) or polynucleotide encoding the same) are provided separately, e.g., in a separate composition. Such compositions can include other chemical or physical agents (e.g., solvents, surfactants, proteins or enzymes, transfection agents, particulates or nanoparticulates), such as those described above as useful in the polynucleotide compositions. For example, porous silica nanoparticles are useful for delivering a DNA recombinase into maize cells; see, e.g., Martin-Ortigosa et al. (2015) Plant Physiol., 164:537-547, and can be adapted to providing a ndRGDBP or polynucleotide encoding the same into a maize or other plant cell. In one embodiment, the polynucleotide composition includes a gRNA and the ndRGDBP, and further includes a surfactant and a cell-penetrating peptide (CPP) which can be operably linked to the ndRGDBP. In an embodiment, the polynucleotide composition includes a plasmid or viral vector that encodes both the gRNA and the ndRGDBP, and further includes a surfactant and carbon nanotubes. In an embodiment, the polynucleotide composition includes multiple gRNAs and an mRNA encoding the ndRGDBP, and further includes particles (e.g., gold or tungsten particles), and the polynucleotide composition is delivered to a plant cell or plant protoplast by Biolistics. In any of the aforementioned embodiments, it is further contemplated that other polynucleotides of interest including genome altering reagents can also be delivered before, during, or after delivery of the gRNA and the ndRGDBP.

In certain embodiments, the plant, plant explant, or plant part from which a plant cell is obtained or isolated is treated with one or more chemical, enzymatic, or physical agent(s) in the process of obtaining, isolating, or treating the plant cell. In certain embodiments, the plant cell, plant, plant explant, or plant part is treated with an abrasive, a caustic agent, a surfactant such as Silwet L-77 or a cationic lipid, or an enzyme such as cellulase. In any of the aforementioned embodiments, it is further contemplated that other polynucleotides of interest including genome altering reagents can also be delivered before, during, or after delivery of the endogenous gene transcription agents.

In certain embodiments, one or more than one chemical, enzymatic, or physical agent, separately or in combination with the polynucleotide composition encoding the exogenous gene transcription agent that stimulates transcription of the endogenous ODP2 gene and/or the endogenous WUS2 gene, is provided/applied at a location in the plant or plant part other than the plant location, part, or tissue from which the plant cell is treated, obtained, or isolated. In certain embodiments, the polynucleotide composition is applied to adjacent or distal cells or tissues and is transported (e.g., through the vascular system or by cell-to-cell movement) to the meristem from which plant cells are subsequently isolated. In certain embodiments, the polynucleotide-containing composition is applied by soaking a seed or seed fragment or zygotic or somatic embryo in the polynucleotide-containing composition, whereby the polynucleotide is delivered to the plant cell. In certain embodiments, a flower bud or shoot tip is contacted with a polynucleotide-containing composition, whereby the polynucleotide is delivered to cells in the flower bud or shoot tip from which desired plant cells (e.g., plant cells subjected to a transient increase in expression of the endogenous ODP2 gene and/or the endogenous WUS2 gene) are obtained. In certain embodiments, a polynucleotide-containing composition is applied to the surface of a plant or of a part of a plant (e.g., a leaf surface), whereby the polynucleotide(s) are delivered to tissues of the plant from which desired plant cells are obtained. In certain embodiments a whole plant or plant tissue is subjected to particle- or nanoparticle-mediated delivery (e.g., Biolistics or carbon nanotube or nanoparticle delivery) of a polynucleotide-containing composition, whereby the polynucleotide(s) are delivered to cells or tissues from which plant cells are subsequently obtained. In any of the aforementioned embodiments, it is further contemplated that other polynucleotides of interest including genome altering reagents can also be delivered before, during, or after delivery of the endogenous gene transcription agents.

Genome altering reagents include gene editing molecules for inducing a genetic modification in the plant cells having improved plant cell regenerative potential provided herein. In certain embodiments, such genome altering reagents can include: (i) a polynucleotide selected from the group consisting of an RNA guide for an RNA-guided nuclease, a DNA encoding an RNA guide for an RNA-guided nuclease; (ii) a nuclease selected from the group consisting of an RNA-guided nuclease, an RNA-guided DNA endonuclease, a type II Cas nuclease, a Cas9, a type V Cas nuclease, a Cpf1, a CasY, a CasX, a C2c1, a C2c3, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), Argonaute, a meganuclease or engineered meganuclease; (iii) a polynucleotide encoding one or more nucleases capable of effectuating site-specific modification of a target nucleotide sequence; and/or (iv) a donor template polynucleotide. In certain embodiments, at least one delivery agent is selected from the group consisting of solvents, fluorocarbons, glycols or polyols, surfactants; primary, secondary, or tertiary amines and quaternary ammonium salts; organosilicone surfactants; lipids, lipoproteins, lipopolysaccharides; acids, bases, caustic agents; peptides, proteins, or enzymes; cell-penetrating peptides; RNase inhibitors; cationic branched or linear polymers; dendrimers; counter-ions, amines or polyamines, osmolytes, buffers, and salts; polynucleotides; transfection agents; antibiotics; chelating agents such as ammonium oxalate, EDTA, EGTA, or cyclohexane diamine tetraacetate, non-specific DNA double-strand-break-inducing agents; and antioxidants; particles or nanoparticles, magnetic particles or nanoparticles, abrasive or scarifying agents, needles or microneedles, matrices, and grids. In certain embodiments, the plant cell, system, method, or composition comprising the plant cells provided herein further includes (a) at least one plant cell having a Cas9, a Cpf1, a CasY, a CasX, a C2c1, or a C2c3 nuclease; (b) at least one guide RNA; and (c) optionally, at least one chemical, enzymatic, or physical delivery agent.

Gene editing molecules of use in the systems, methods, compositions, and reaction mixtures provided herein include molecules capable of introducing a double-strand break (“DSB”) in double-stranded DNA, such as in genomic DNA or in a target gene located within the genomic DNA as well as accompanying guide RNA or donor template polynucleotides. Examples of such gene editing molecules include: (a) a nuclease selected from the group consisting of an RNA-guided nuclease, an RNA-guided DNA endonuclease, a type II Cas nuclease, a Cas9, a type V Cas nuclease, a Cpf1, a CasY, a CasX, a C2c1, a C2c3, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TAL-effector nuclease), an Argonaute, and a meganuclease or engineered meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effectuating site-specific alteration (such as introduction of a DSB) of a target nucleotide sequence; (c) a guide RNA (gRNA) for an RNA-guided nuclease, or a DNA encoding a gRNA for an RNA-guided nuclease; and (d) donor template polynucleotides.

CRISPR-type genome editing can be adapted for use in the plant cells, systems, methods, and compositions provided herein in several ways. CRISPR elements, i.e., gene editing molecules comprising CRISPR endonucleases and CRISPR single-guide RNAs or polynucleotides encoding the same, are useful in effectuating genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny. In certain embodiments, the CRISPR elements are provided directly to the plant cells, systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of a cell free synthetic process (e.g., in vitro translation), or as isolated or semi-purified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, genome-inserted CRISPR elements are useful in plant lines adapted for use in the systems, methods, and compositions provide herein. In certain embodiments, plants or plant cells used in the systems, methods, and compositions provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Cas9, a Cpf1-type or other CRISPR endonuclease). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 A1, which is incorporated herein by reference for its disclosure of DNA encoding Cpf1 endonucleases and guide RNAs and PAM sites. Introduction of one or more of a wide variety of CRISPR guide RNAs that interact with CRISPR endonucleases integrated into a plant genome or otherwise provided to a plant is useful for genetic editing for providing desired phenotypes or traits, for trait screening, or for gene editing mediated trait introgression (e.g., for introducing a trait into a new genotype without backcrossing to a recurrent parent or with limited backcrossing to a recurrent parent). Multiple endonucleases can be provided in expression cassettes with the appropriate promoters to allow multiple genome editing in a spatially or temporally separated fashion in either in chromosome DNA or episome DNA.

CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016/0138008A1 and US2015/0344912A1, and in U.S. Pat. Nos. 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 A1. Other CRISPR nucleases useful for editing genomes include C2c1 and C2c3 (see Shmakov et al. (2015) Mol. Cell, 60:385-397) and CasX and CasY (see Burstein et al. (2016) Nature, doi:10.1038/nature21059). Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in International Patent Application PCT/US2015/018104 (published as WO 2015/131101 and claiming priority to U.S. Provisional Patent Application 61/945,700). Methods of using CRISPR technology for genome editing in plants are disclosed in US Patent Application Publications US 2015/0082478A1 and US 2015/0059010A1 and in International Patent Application PCT/US2015/038767 A1 (published as WO 2016/007347 and claiming priority to U.S. Provisional Patent Application 62/023,246). All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.

For the purposes of gene editing, CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308. At least 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavage to occur; for Cpf1 at least 16 nucleotides of gRNA sequence are needed to achieve detectable DNA cleavage and at least 18 nucleotides of gRNA sequence were reported necessary for efficient DNA cleavage in vitro; see Zetsche et al. (2015) Cell, 163:759-771. In practice, guide RNA sequences are generally designed to have a length of 17-24 nucleotides (frequently 19, 20, or 21 nucleotides) and exact complementarity (i.e., perfect base-pairing) to the targeted gene or nucleic acid sequence; guide RNAs having less than 100% complementarity to the target sequence can be used (e.g., a gRNA with a length of 20 nucleotides and 1-4 mismatches to the target sequence) but can increase the potential for off-target effects. The design of effective guide RNAs for use in plant genome editing is disclosed in US Patent Application Publication 2015/0082478 A1, the entire specification of which is incorporated herein by reference. More recently, efficient gene editing has been achieved using a chimeric “single guide RNA” (“sgRNA”), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing); see, for example, Cong et al. (2013) Science, 339:819-823; Xing et al. (2014) BMC Plant Biol., 14:327-340. Chemically modified sgRNAs have been demonstrated to be effective in genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. The design of effective gRNAs for use in plant genome editing is disclosed in US Patent Application Publication 2015/0082478 A1, the entire specification of which is incorporated herein by reference.

Other nucleases capable of effecting site-specific modification of a target nucleotide sequence in the systems, methods, and compositions provided herein include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TAL-effector nucleases or TALENs), Argonaute proteins, and a meganuclease or engineered meganuclease. Zinc finger nucleases (ZFNs) are engineered proteins comprising a zinc finger DNA-binding domain fused to a nucleic acid cleavage domain, e.g., a nuclease. The zinc finger binding domains provide specificity and can be engineered to specifically recognize any desired target DNA sequence. For a review of the construction and use of ZFNs in plants and other organisms, see, e.g., Urnov et al. (2010) Nature Rev. Genet., 11:636-646. The zinc finger DNA binding domains are derived from the DNA-binding domain of a large class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains a tandem array of at least three zinc “fingers” each recognizing a specific triplet of DNA. A number of strategies can be used to design the binding specificity of the zinc finger binding domain. One approach, termed “modular assembly”, relies on the functional autonomy of individual zinc fingers with DNA. In this approach, a given sequence is targeted by identifying zinc fingers for each component triplet in the sequence and linking them into a multifinger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to accommodate the ability of zinc fingers to contact neighboring fingers as well as nucleotide bases outside their target triplet. Typically, the engineered zinc finger DNA binding domain has a novel binding specificity, compared to a naturally-occurring zinc finger protein. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of databases of triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, e.g., U.S. Pat. Nos. 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) are well known and described in the literature. In addition, enhancement of binding specificity for zinc finger binding domains has been described in U.S. Pat. No. 6,794,136, incorporated herein by reference in its entirety. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. Examples of linker sequences are publicly known, e.g., see U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949, incorporated herein by reference in their entirety. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fok1. This endonuclease must dimerize to cleave DNA. Thus, cleavage by Fok1 as part of a ZFN requires two adjacent and independent binding events, which must occur in both the correct orientation and with appropriate spacing to permit dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites. Fok1 variants with enhanced activities have been described; see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96-107.

Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to a non-specific DNA cleavage domain to prepare genome editing proteins, referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fok1, can be conveniently used. For a description of the use of TALENs in plants, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA, 108:2623-2628 and Mahfouz (2011) GM Crops, 2:99-103.

Argonautes are proteins that can function as sequence-specific endonucleases by binding a polynucleotide (e.g., a single-stranded DNA or single-stranded RNA) that includes sequence complementary to a target nucleotide sequence) that guides the Argonaut to the target nucleotide sequence and effects site-specific alteration of the target nucleotide sequence; see, e.g., US Patent Application Publication 2015/0089681, incorporated herein by reference in its entirety.

In related embodiments, zinc finger nucleases, TALENs, and Argonautes are used in conjunction with other functional domains. For example, the nuclease activity of these nucleic acid targeting systems can be altered so that the enzyme binds to but does not cleave the DNA. Examples of functional domains include transposase domains, integrase domains, recombinase domains, resolvase domains, invertase domains, protease domains, DNA methyltransferase domains, DNA hydroxylmethylase domains, DNA demethylase domains, histone acetylase domains, histone deacetylase domains, nuclease domains, repressor domains, activator domains, nuclear-localization signal domains, transcription-regulatory protein (or transcription complex recruiting) domains, cellular uptake activity associated domains, nucleic acid binding domains, antibody presentation domains, histone modifying enzymes, recruiter of histone modifying enzymes; inhibitor of histone modifying enzymes, histone methyltransferases, histone demethylases, histone kinases, histone phosphatases, histone ribosylases, histone deribosylases, histone ubiquitinases, histone deubiquitinases, histone biotinases and histone tail proteases. Non-limiting examples of functional domains include a transcriptional activation domain, a transcription repression domain, and an SHH1, SUVH2, or SUVH9 polypeptide capable of reducing expression of a target nucleotide sequence via epigenetic modification; see, e.g., US Patent Application Publication 2016/0017348, incorporated herein by reference in its entirety. Genomic DNA may also be modified via base editing using a fusion between a catalytically inactive Cas9 (dCas9) fused to a cytidine deaminase which converts cytosine (C) to uridine (U), thereby effecting a C to T substitution; see Komor et al. (2016) Nature, 533:420-424. In other embodiments, adenine base editors (ABEs) can be used to convert A/T base pairs to G/C base pairs in genomic DNA (Gaudelli et al., 2017).

Other genome altering reagents used in plant cells and methods provided herein include transgenes or vectors comprising the same. Such transgenes can confer useful traits that include herbicide tolerance, pest tolerance (e.g., tolerance to insects, nematodes, or plant pathogenic fungi and bacteria), improved yield, increased and/or qualitatively improved oil, starch, and protein content, improved abiotic stress tolerance (e.g., improved or enhanced water use efficiency or drought tolerance, osmotic stress tolerance, high salinity stress tolerance, heat stress tolerance, enhanced cold tolerance, including cold germination tolerance), and the like. Such transgenes include both transgenes that confer the trait by expression of an exogenous protein as well as transgenes that confer the trait by inhibiting expression of endogenous plant genes (e.g., by inducing an siRNA response which inhibits expression of the endogenous plant genes). Transgenes that can provide such traits are disclosed in US Patent Application Publication Nos. 20170121722 and 20170275636, which are each incorporated herein by reference in their entireties and specifically with respect to such disclosures.

In some embodiments, one or more polynucleotides or vectors driving expression of one or more polynucleotides encoding any of the aforementioned exogenous gene transcription agents and/or genome altering reagents are introduced into a plant cell. In certain embodiments, a polynucleotide vector comprises a regulatory element such as a promoter operably linked to one or more polynucleotides encoding exogenous gene transcription agents or genome altering reagents. In such embodiments, expression of these polynucleotides can be controlled by selection of the appropriate promoter, particularly promoters functional in a plant cell; useful promoters include constitutive, conditional, inducible, and temporally or spatially specific promoters (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter). Developmentally regulated promoters that can be used include Phospholipid Transfer Protein (PLTP), fructose-1,6-bisphosphatase protein, NAD(P)-binding Rossmann-Fold protein, adipocyte plasma membrane-associated protein-like protein, Rieske [2Fe-2S] iron-sulfur domain protein, chlororespiratory reduction 6 protein, D-glycerate 3-kinase, chloroplastic-like protein, chlorophyll a-b binding protein 7, chloroplastic-like protein, ultraviolet-B-repressible protein, Soul heme-binding family protein, Photosystem I reaction center subunit psi-N protein, and short-chain dehydrogenase/reductase protein that are disclosed in US Patent Application Publication No. 20170121722, which is incorporated herein by reference in its entirety and specifically with respect to such disclosure. In certain embodiments, the promoter is operably linked to nucleotide sequences encoding multiple guide RNAs, wherein the sequences encoding guide RNAs are separated by a cleavage site such as a nucleotide sequence encoding a microRNA recognition/cleavage site or a self-cleaving ribozyme (see, e.g., Ferré-D'Amaré and Scott (2014) Cold Spring Harbor Perspectives Biol., 2:a003574). In certain embodiments, the promoter is an RNA polymerase III promoter operably linked to a nucleotide sequence encoding one or more guide RNAs. In certain embodiments, the promoter operably linked to one or more polynucleotides is a constitutive promoter that drives gene expression in plant cells. In certain embodiments, the promoter drives gene expression in the nucleus or in an organelle such as a chloroplast or mitochondrion. Examples of constitutive promoters include a CaMV 35S promoter as disclosed in U.S. Pat. Nos. 5,858,742 and 5,322,938, a rice actin promoter as disclosed in U.S. Pat. No. 5,641,876, a maize chloroplast aldolase promoter as disclosed in U.S. Pat. No. 7,151,204, and the nopaline synthase (NOS) and octopine synthase (OCS) promoters from Agrobacterium tumefaciens. In certain embodiments, the promoter operably linked to one or more polynucleotides encoding elements of a genome-editing system is a promoter from figwort mosaic virus (FMV), a RUBISCO promoter, or a pyruvate phosphate dikinase (PPDK) promoter, which is active in photosynthetic tissues. Other contemplated promoters include cell-specific or tissue-specific or developmentally regulated promoters, for example, a promoter that limits the expression of the nucleic acid targeting system to germline or reproductive cells (e.g., promoters of genes encoding DNA ligases, recombinases, replicases, or other genes specifically expressed in germline or reproductive cells). In certain embodiments, the genome alteration is limited only to those cells from which DNA is inherited in subsequent generations, which is advantageous where it is desirable that expression of the genome-editing system be limited in order to avoid genotoxicity or other unwanted effects. All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety.

Expression vectors or polynucleotides provided herein may contain a DNA segment near the 3′ end of an expression cassette that acts as a signal to terminate transcription and directs polyadenylation of the resultant mRNA, and may also support promoter activity. Such a 3′ element is commonly referred to as a “3′-untranslated region” or “3′-UTR” or a “polyadenylation signal.” In some cases, plant gene-based 3′ elements (or terminators) consist of both the 3′-UTR and downstream non-transcribed sequence (Nuccio et al., 2015). Useful 3′ elements include: Agrobacterium tumefaciens nos 3′, tml 3′, tmr 3′, tms 3′, ocs 3′, and tr7 3′ elements disclosed in U.S. Pat. No. 6,090,627, incorporated herein by reference, and 3′ elements from plant genes such as the heat shock protein 17, ubiquitin, and fructose-1,6-biphosphatase genes from wheat (Trilicum aestivum), and the glutelin, lactate dehydrogenase, and beta-tubulin genes from rice (Oryza sativa), disclosed in US Patent Application Publication 2002/0192813 A1, incorporated herein by reference.

In certain embodiments, a vector or polynucleotide comprising an expression cassette includes additional components, e.g., a polynucleotide encoding a drug resistance or herbicide gene or a polynucleotide encoding a detectable marker such as green fluorescent protein (GFP) or beta-glucuronidase (gus) to allow convenient screening or selection of cells expressing the vector or polynucleotide. Selectable markers include genes that confer resistance to herbicidal compounds, such as glyphosate, sulfonylureas, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Since the transient expression of endogenous ODP2 and/or WUS2 genes can accelerate somatic embryogenesis and embryo maturation, selectable marker genes, selective agents, and conditions can be adjusted to minimize formation of un-edited or untransformed regenerable plant structures (e.g., “escapes”). Such selectable marker genes and selective agents include the maize HRA gene (Lee et al., 1988, EMBO J 7:1241-1248) which confers resistance to sulfonylureas and imidazolinones, the CP4 gene that confers resistance to glyphosate (U.S. Reissue Pat. RE39,247, specifically incorporated herein by reference in its entirety and with respect to such genes and related selection methods), the GAT gene which confers resistance to glyphosate (Castle et al., 2004, Science 304:1151-1154), genes that confer resistance to spectinomycin such as the aadA gene (Svab et al., 1990, Plant Mol Biol. 14:197-205) and the bar gene that confers resistance to glufosinate ammonium (White et al., 1990, Nucl. Acids Res. 25:1062), and PAT (or moPAT for corn, see Rasco-Gaunt et al., 2003, Plant Cell Rep. 21:569-76; also see Sivamani et al., 2019) and the PMI gene that permits growth on mannose-containing medium (Negrotto et al., 2000, Plant Cell Rep. 22:684-690).

EMBODIMENTS

Various embodiments of the plant cells and methods provided herein are included in the following non-limiting list of embodiments.

-   -   1. A plant cell wherein expression of an endogenous ODP2         polypeptide and/or expression of an endogenous WUS2 polypeptide         is increased in comparison to the expression of the endogenous         ODP2 and/or the endogenous WUS2 polypeptides in a control plant         cell, wherein the plant cell can form a regenerable plant         structure, and optionally wherein the plant cell is a monocot         plant cell or optionally wherein the plant cell is a maize,         wheat, sorghum, or rice plant cell.     -   2. The plant cell of embodiment 1, wherein an exogenous         polynucleotide encoding an ODP2 and/or WUS2 polypeptide is         absent before and/or during the increase in expression of the         endogenous ODP2 polypeptide and/or the endogenous WUS2         polypeptide.     -   3. The plant cell of embodiment 1 or 2, wherein the increase in         expression of the endogenous ODP2 polypeptide and/or expression         of the endogenous WUS2 polypeptide is sufficient to increase in         proliferation, somatic embryogenesis, and/or regeneration         capacity of the plant cell in comparison to the control plant         cell.     -   4. The plant cell of any one of embodiments 1, 2, or 3, wherein         the increase in expression of the endogenous ODP2 polypeptide         and/or expression of the endogenous WUS2 polypeptide is         sufficient to increase in transformation efficiency and/or         endogenous gene editing efficiency of the plant cell in         comparison to the control plant cell.     -   5. The plant cell of any one of embodiments 1-3, or 4, wherein         the increase in the expression of the endogenous ODP2         polypeptide and/or expression of the endogenous WUS2 polypeptide         is for a period of about 12, 24, 30, or 36 hours to about 168 or         192 hours.     -   6. The plant cell of any one of embodiments 1-4, or 5, wherein         said cell is located within or obtained from a cultured plant         tissue explant, an immature embryo, a mature embryo, a leaf,         and/or callus.     -   7. The plant cell of embodiment 6, wherein the plant tissue         explant, embryo, or callus exhibits an increase in         proliferation, somatic embryogenesis, and/or regeneration         capacity in comparison to the control plant tissue explant,         embryo, or callus which was not subjected to an increase in         expression of the endogenous ODP2 polypeptide and/or the         endogenous WUS2 polypeptide.     -   8. The plant cell of embodiment 6, wherein the plant tissue         explant, embryo, or callus exhibits an increase in         transformation efficiency and/or endogenous gene editing         efficiency in comparison to the control plant tissue explant,         embryo, or callus which was not subjected to an increase in         expression of the endogenous ODP2 polypeptide and/or the         endogenous WUS2 polypeptide.     -   9. The plant cell of any one of embodiments 1 to 8 wherein the         plant cell, plant tissue explant, embryo, or callus comprises         inbred germplasm, haploid germplasm, and/or a         regeneration-recalcitrant germplasm.     -   10. The plant cell of any one of embodiments 1 to 8, wherein the         plant cell is derived from the L1 or L2 layer of an immature or         mature embryo.     -   11. The plant cell of any one of embodiments 1 to 8, wherein the         plant cell is a maize plant cell and the ODP2 polypeptide         comprises an amino acid sequence having at least 95%, 96%, 97%,         or 99% amino acid sequence identity across the entire length of         SEQ ID NO:1.     -   12. The plant cell of embodiment 11, wherein the endogenous ODP2         polypeptide is encoded by the endogenous maize ODP2 gene located         on maize chromosome 3 and/or that is encoded by an endogenous         polynucleotide that is operably linked to an endogenous maize         ODP2 promoter of SEQ ID NO:3, SEQ ID NO:71, or an allelic         variant thereof.     -   13. The plant cell of any one of embodiments 1 to 12, wherein         the plant cell is a maize plant cell and the endogenous WUS2         polypeptide comprises an amino acid sequence having at least         95%, 96%, 97%, or 99% amino acid sequence identity across the         entire length of SEQ ID NO:2.     -   14. The plant cell of embodiment 13, wherein the endogenous WUS2         polypeptide is encoded by the endogenous maize WUS2 gene located         on maize chromosome 10 and/or that is encoded by an endogenous         polynucleotide that is operably linked to an endogenous maize         WUS2 promoter of SEQ ID NO:4 or an allelic variant thereof.     -   15. The plant cell of any one of embodiments 1 to 14, wherein         the expression of the endogenous ODP2 polypeptide and/or the         endogenous WUS2 polypeptide is transiently increased with at         least one exogenous gene transcription agent that stimulates         transcription of the endogenous ODP2 gene and/or with at least         one exogenous gene transcription agent that stimulates         transcription of the endogenous WUS2 gene.     -   16. The plant cell of embodiment 15, wherein the exogenous gene         transcription agent is provided as: (i) an exogenous protein;         or (ii) as an exogenous protein and an exogenous guide RNA.     -   17. The plant cell of embodiment 16, wherein the exogenous         protein of either (i) or (ii) is provided in the cell in the         absence of an exogenous polynucleotide that encodes the protein.     -   18. The plant cell of embodiment 16, wherein the exogenous         protein of either (i) or (ii) is provided in the cell by an         exogenous polynucleotide comprising a promoter that is operably         linked to a polynucleotide that encodes the protein or by an         exogenous RNA molecule that encodes the protein.     -   19. The plant cell of embodiment 18, wherein the exogenous RNA         molecule that encodes the protein comprises an mRNA or an RNA         with an internal ribosome entry site (IRES).     -   20. The plant cell of embodiment 19, wherein the exogenous         polynucleotide or exogenous RNA are operably linked to a         polynucleotide comprising a viral vector or T-DNA in the cell.     -   21. The plant cell of embodiment 20, wherein the exogenous         protein, exogenous polynucleotide, and/or exogenous guide RNA         are in part associated with an exogenous particle within the         cell.     -   22. The plant cell of embodiment 15, wherein the exogenous gene         transcription agent comprises: (i) a domain or complex which         binds to the promoter or 5′ untranslated region (5′ UTR) of the         endogenous ODP2 gene or to the promoter or 5′ UTR of the         endogenous WUS2 gene; and (ii) a transcription activation         domain, wherein the transcription activation domain is operably         linked or operably associated with the domain or complex.     -   23. The plant cell of embodiment 22, wherein the exogenous gene         transcription agent further comprises an operably linked nuclear         localization signal (NLS).     -   24. The plant cell of embodiment 22, wherein the domain that         binds the promoter or 5′ UTR comprises an artificial zinc finger         (AZF) DNA binding domain polypeptide or an artificial         transcription activator-like effector (TALE) DNA binding         polypeptide.     -   25. The plant cell of embodiment 24, wherein the plant cell is a         maize plant cell and the artificial zinc finger DNA binding         domain that binds the ODP2 promoter comprises: (i) a polypeptide         having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence         identity across the entire length of SEQ ID NO: 5 or 8; or (ii)         a polypeptide having at one or more conservative and/or         semi-conservative amino acid substitutions in SEQ ID NO: 5 or 8.     -   26. The plant cell of embodiment 24, wherein the plant cell is a         maize plant cell and the artificial zinc finger DNA binding         domain that binds the WUS2 promoter comprises: (i) a polypeptide         having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence         identity across the entire length of SEQ ID NO: 11 or 14; or         comprises a polypeptide having at one or more conservative         and/or semi-conservative amino acid substitutions in SEQ ID NO:         11 or 14.     -   27. The plant cell of embodiment 24, wherein the plant cell is a         maize plant cell and the artificial TALE DNA binding polypeptide         that binds the ODP2 promoter comprises: (i) a polypeptide having         at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity         across the entire length of SEQ ID NO: 27, 81, 84, or 87;         or (ii) a polypeptide having at one or more conservative and/or         semi-conservative amino acid substitutions in SEQ ID NO: 23, 25,         27, 81, 84, or 87.     -   28. The plant cell of embodiment 24, wherein the plant cell is a         maize plant cell and the artificial TALE DNA binding domain that         binds the WUS2 promoter comprises: (i) a polypeptide having at         least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across         the entire length of SEQ ID NO: 17, 19, 21, 72, 75, or 78;         or (ii) a polypeptide having at one or more conservative and/or         semi-conservative amino acid substitutions in SEQ ID NO: 17, 19,         21, 72, 75, or 78.     -   29. The plant cell of embodiment 22, wherein the complex that         binds the promoter or 5′ UTR comprises: (i) an RNA guided DNA         binding polypeptide that is nuclease activity deficient and a         guide RNA comprising about an 18 or 19 to about a 21 or 22         nucleotide polynucleotide sequence which is complementary to a         sequence immediately adjacent to a protospacer adjacent motif         (PAM) in the promoter or 5′ UTR; or (ii) a 20, 21, 22, 23, or 24         nucleotide polynucleotide sequence which is complementary to a         sequence immediately adjacent to a protospacer adjacent motif         (PAM) in the promoter or 5′ UTR.     -   30. The plant cell of embodiment 29, wherein the RNA guided DNA         binding polypeptide comprises a dCAS9 polypeptide, comprises a         polypeptide having at least 85%, 90%, 95%, 97%, 98%, or 99%         sequence identity across the entire length of SEQ ID NO: 29, or         comprises a polypeptide having at one or more conservative         and/or semi-conservative amino acid substitutions in SEQ ID NO:         29.     -   31. The plant cell of embodiment 29, wherein the RNA guided DNA         binding polypeptide comprises; (i) a dCpf1 polypeptide;     -   (ii) a polypeptide having one or more conservative and/or         semi-conservative amino acid substitutions in SEQ ID NO: 44 or         at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity         across the entire length of SEQ ID NO: 44 and a D917A, E1006A,         E1028A, D1255A, and/or N1257A amino acid substitution;     -   (iii) a polypeptide having one or more conservative and/or         semi-conservative amino acid substitutions in SEQ ID NO: 45 or         at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity         across the entire length of SEQ ID NO: 45 and a D832A, E925A,         and/or D1148A amino acid substitution;     -   (iv) a polypeptide having one or more conservative and/or         semi-conservative amino acid substitutions in SEQ ID NO: 46 or         at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity         across the entire length of SEQ ID NO: 46 and a D917A, E1006A,         E1028A, D1255A, and/or N1257A amino acid substitution; or     -   (v) a polypeptide having one or more conservative and/or         semi-conservative amino acid substitutions in SEQ ID NO: 47 or         at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity         across the entire length of SEQ ID NO: 47 and a D901A and/or         E1228A amino acid substitution.     -   32. The plant cell of embodiment 29, wherein the plant cell is a         maize plant cell and the guide RNA comprises a sequence that is         complementary to either strand of an ODP2 promoter comprising         SEQ ID NO: 3 or SEQ ID NO: 71 or to either strand of an WUS2         promoter comprising SEQ ID NO: 4, wherein the complementary         sequence is immediately adjacent to a protospacer adjacent motif         (PAM) in SEQ ID NO: 3, 71, or 4.     -   33. The plant cell of embodiment 32, wherein the guide RNA         comprises an RNA encoded by SEQ ID NO: 31, 32, 33, 34, or 35         that is complementary to a sequence in the endogenous maize ODP2         promoter.     -   34. The plant cell of embodiment 32, wherein the guide RNA         comprises an RNA encoded by SEQ ID NO: 36, 37, 38, 39, or 40         that is complementary to a sequence in the endogenous maize WUS2         promoter.     -   35. The plant cell of any one of embodiments 1 to 34, wherein         the cell comprises at least two exogenous gene transcription         agents that stimulate transcription of the endogenous ODP2 gene         and/or at least two exogenous gene transcription agents that         stimulate transcription of the endogenous WUS2 gene.     -   36. The plant cell of embodiment 35, wherein the exogenous gene         transcription agents comprise a transcriptional activation         domain that is operably linked to or operably associated         with: (i) an artificial zinc finger (AZF) DNA binding domain         polypeptide; (ii) an artificial TALE DNA binding         polypeptide; (iii) an RNA guided DNA binding polypeptide that is         nuclease activity deficient and a guide RNA, or any combination         thereof.     -   37. The plant cell of embodiment 35, wherein the plant cell is a         maize plant cell and the exogenous gene transcription agents         that stimulate transcription of the endogenous WUS2 gene         comprise a combination of at least two artificial TALE         transcription factors comprising a transcriptional activation         domain and an artificial TALE DNA binding polypeptide of SEQ ID         NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:72, SEQ ID NO:75,         and SEQ ID NO:78.     -   38. The plant cell of embodiment 35, wherein the plant cell is a         maize plant cell and the exogenous gene transcription agents         that stimulate transcription of the endogenous ODP2 gene         comprise a combination of at least two distinct artificial TALE         transcription factors that each comprise a transcriptional         activation domain that is operably linked to or operably         associated with an artificial TALE DNA sequence recognition         domain polypeptide of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27,         SEQ ID NO:81, SEQ ID NO:84, or SEQ ID NO:87.     -   39. The plant cell of any one of embodiments 1 to 38, wherein         the cell further comprises a genome altering reagent.     -   40. The plant cell of any one of embodiments 1 to 39, wherein         the regenerable plant structure comprises a somatic embryo,         embryogenic callus, somatic meristem, organogenic callus, a         shoot, or a shoot further comprising roots.     -   41. A method of producing a regenerable plant structure,         comprising:         -   (i) introducing into a plant cell at least one exogenous             gene transcription agent which transiently increases             expression of an endogenous ODP2 polypeptide and/or at least             one exogenous gene transcription agent which transiently             increases expression of an endogenous WUS2 polypeptide,             wherein the expression is increased in comparison to the             expression of the endogenous ODP2 and/or the endogenous WUS2             polypeptides in a control plant cell; and,         -   (ii) culturing the plant cell to produce a regenerable plant             structure;         -   optionally wherein the plant cell is a monocot plant cell or             optionally wherein the monocot plant cell is a maize, wheat,             sorghum, or rice plant cell.     -   42. The method of embodiment 41, wherein an exogenous         polynucleotide encoding an ODP2 and/or WUS2 polypeptide is         absent before and/or during the transient increase in expression         of the endogenous ODP2 polypeptide and/or the endogenous WUS2         polypeptide.     -   43. The method of embodiment 41 or 42, wherein the transient         increase in expression of the endogenous ODP2 polypeptide and/or         expression of the endogenous WUS2 polypeptide is sufficient to         increase proliferation, somatic embryogenesis, and/or         regeneration capacity of the plant cell in comparison to the         control plant cell.     -   44. The method of any one of embodiments 41 to 43, wherein the         transient increase in expression of the endogenous ODP2         polypeptide and/or expression of the endogenous WUS2 polypeptide         is sufficient to increase transformation efficiency and/or         endogenous gene editing efficiency in comparison to the control         plant cell.     -   45. The method of any one of embodiments 41 to 44, wherein the         transient increase in the expression of the endogenous ODP2         polypeptide and/or expression of the endogenous WUS2 polypeptide         is for a period of about 24, 30, or 36 hours to about 168 or 192         hours.     -   46. The method of any one of embodiments 41 to 45, wherein the         exogenous gene transcription agent is introduced by         electroporation, particle bombardment, transfection,         Agrobacterium-mediated transformation or viral vector-mediated         transfer.     -   47. The method of embodiment 41, wherein the exogenous gene         transcription agent is introduced as: (i) an exogenous protein;         or (ii) as an exogenous protein and an exogenous guide RNA,         wherein said protein and said guide RNA are optionally complexed         as a RNP.     -   48. The method of embodiment 47, wherein the exogenous protein         of either (i) or (ii) is introduced in the cell in the absence         of an exogenous polynucleotide that encodes the protein.     -   49. The method of embodiment 47, wherein the exogenous protein         of either (i) or (ii) is introduced in the cell by an exogenous         polynucleotide comprising a promoter that is operably linked to         a polynucleotide that encodes the protein or an exogenous RNA         molecule that encodes the protein.     -   50. The method of embodiment 49, wherein the exogenous         polynucleotide or exogenous RNA are operably linked to a         polynucleotide comprising a viral vector or T-DNA in the cell.     -   51. The method of embodiment 49, wherein the exogenous         polynucleotide is not integrated into the nuclear or plastid         genome of the plant cell.     -   52. The method of any one of embodiments 41 to 51, wherein said         plant cell is located within or obtained from a cultured plant         tissue explant, an immature embryo, a mature embryo, a leaf,         and/or callus.     -   53. The method of embodiment 52, wherein the plant cell is         derived from the L1 or L2 layer of an immature or mature embryo.     -   54. The method of any one of embodiments 41 to 53, wherein the         plant cell, plant tissue explant, embryo, or callus comprises         inbred germplasm, haploid germplasm, and/or a         regeneration-recalcitrant germplasm.     -   55. The method of any one of embodiments 41 to 54, wherein the         regenerable plant structure comprises a somatic embryo,         embryogenic callus, somatic meristem, organogenic callus, a         shoot, or a shoot further comprising roots.     -   56. The method of any one of embodiments 41 to 55, wherein the         culturing comprises growing of the plant cell in plant cell         growth media comprising an auxin concentration sufficient to         induce formation of a somatic embryo, embryogenic callus,         somatic meristem, and/or organogenic callus.     -   57. The method of embodiment 56, wherein the culturing further         comprises growing the somatic embryo, embryogenic callus,         somatic meristem, and/or organogenic callus in plant cell growth         media comprising concentrations of auxin and cytokinin         sufficient to induce formation of a shoot.     -   58. The method of embodiment 57, wherein the culturing further         comprises growing the shoot in a plant cell growth media until         the shoot forms roots.     -   59. The method of any one of embodiments 41 to 58, further         comprising the step of introducing a genome altering reagent         into the cell at step (i), (ii), or (i) and (ii).     -   60. The method of embodiment 59, wherein the genome altering         reagent comprises a transgene, a vector comprising a transgene,         or genome editing molecules.     -   61. The method of embodiment 60, wherein the vector comprises a         T-DNA or viral vector.     -   62. The method of embodiment 59, wherein the genome editing         molecules comprise an RNA-guided nuclease or a polynucleotide         encoding an RNA-guided nuclease, a guide RNA or a polynucleotide         encoding a guide RNA, and optionally a donor template         polynucleotide or a polynucleotide encoding a donor template         polynucleotide.     -   63. The method of embodiment 62, wherein the RNA-guided nuclease         and guide RNA are introduced as a ribonucleoprotein (RNP)         complex.     -   64. The method of embodiment 59, wherein the genome editing         molecules comprise a transcription activator-like nuclease         (TALEN) protein or a polynucleotide encoding a TALEN protein and         optionally a donor template polynucleotide or a polynucleotide         encoding a donor template polynucleotide.     -   65. The method of embodiment 59, wherein the genome editing         molecules comprise a zinc finger nuclease (ZnfN) protein or a         polynucleotide encoding a ZnfN protein and optionally a donor         template polynucleotide or a polynucleotide encoding a donor         template polynucleotide.     -   66. The method of any one of embodiments 41 to 65, wherein the         plant cell is a maize plant cell and the ODP2 polypeptide         comprises an amino acid sequence having at least 95%, 96%, 97%,         or 99% amino acid sequence identity across the entire length of         SEQ ID NO:1.     -   67. The method of embodiment 66, wherein the endogenous ODP2         polypeptide is encoded by the endogenous maize ODP2 gene located         on maize chromosome 3 and/or that is encoded by an endogenous         polynucleotide that is operably linked to an endogenous maize         ODP2 promoter of SEQ ID NO:3, SEQ ID NO:71, or an allelic         variant thereof.     -   68. The method of any one of embodiments 41 to 67, wherein the         plant cell is a maize plant cell and the endogenous WUS2         polypeptide comprises an amino acid sequence having at least         95%, 96%, 97%, or 99% amino acid sequence identity across the         entire length of SEQ ID NO:2.     -   69. The method of embodiment 68, wherein the endogenous WUS2         polypeptide is encoded by the endogenous maize WUS2 gene located         on maize chromosome 10 and/or that is encoded by an endogenous         polynucleotide that is operably linked to an endogenous maize         WUS2 promoter of SEQ ID NO:4 or an allelic variant thereof.     -   70. The method of any one of embodiments 41 to 69, wherein the         exogenous gene transcription agent is introduced as: (i) an         exogenous protein or exogenous polynucleotide encoding the         protein; or (ii) as an exogenous protein or exogenous         polynucleotide encoding the protein and an exogenous guide RNA.     -   71. The method of embodiment 70, wherein the exogenous protein         of either (i) or (ii) is introduced into the cell in the absence         of an exogenous polynucleotide that encodes the protein.     -   72. The method of embodiment 71, wherein the exogenous protein         of either (i) or (ii) is introduced into the cell by an         exogenous polynucleotide comprising a promoter that is operably         linked to a polynucleotide that encodes the protein or by an         exogenous RNA molecule that encodes the protein.     -   73. The method of embodiment 72, wherein the exogenous RNA         molecule that encodes the protein comprises an mRNA or an RNA         with an internal ribosome entry site (IRES).     -   74. The method of embodiment 72, wherein the exogenous         polynucleotide or exogenous RNA are operably linked to a         polynucleotide comprising a viral vector or T-DNA and wherein         the polynucleotide comprising a viral vector or T-DNA is         introduced into the cell.     -   75. The method of embodiment 70, wherein the exogenous protein,         exogenous polynucleotide, and/or exogenous guide RNA are         introduced into the cell by particle bombardment of the cell.     -   76. The method of any one of embodiments 41 to 75, wherein the         exogenous gene transcription agent comprises: (i) a domain or         complex which binds to the promoter or 5′ untranslated region         (5′ UTR) of the endogenous ODP2 gene or to the promoter or 5′         UTR of the endogenous WUS2 gene; and (ii) a transcription         activation domain, wherein the transcription activation domain         is operably linked or operably associated with the domain or         complex.     -   77. The method of embodiment 76, wherein the exogenous gene         transcription agent further comprises an operably linked nuclear         localization signal (NLS).     -   78. The method of embodiment 76, wherein the domain that binds         the promoter or 5′ UTR comprises an artificial zinc finger (AZF)         DNA binding domain polypeptide or an artificial transcription         activator-like effector (TALE) DNA binding polypeptide.     -   79. The method of embodiment 78, wherein the artificial zinc         finger DNA binding domain that binds the ODP2 promoter         comprises: (i) a polypeptide having at least 85%, 90%, 95%, 97%,         98%, or 99% sequence identity across the entire length of SEQ ID         NO: 5 or 8; or (ii) a polypeptide having at one or more         conservative and/or semi-conservative amino acid substitutions         in SEQ ID NO: 5 or 8.     -   80. The method of embodiment 78, wherein the artificial zinc         finger DNA binding domain that binds the WUS2 promoter         comprises: (i) a polypeptide having at least 85%, 90%, 95%, 97%,         98%, or 99% sequence identity across the entire length of SEQ ID         NO: 11 or 14; or comprises a polypeptide having at one or more         conservative and/or semi-conservative amino acid substitutions         in SEQ ID NO: 11 or 14.     -   81. The method of embodiment 78, wherein the artificial TALE DNA         binding polypeptide that binds the ODP2 promoter comprises: (i)         a polypeptide having at least 85%, 90%, 95%, 97%, 98%, or 99%         sequence identity across the entire length of SEQ ID NO: 23, 25,         27, 81, 84, or 87; or (ii) a polypeptide having at one or more         conservative and/or semi-conservative amino acid substitutions         in SEQ ID NO: 23, 25, 27, 81, 84, or 87.     -   82. The method of embodiment 78, wherein the artificial TALE DNA         binding domain that binds the WUS2 promoter comprises: (i) a         polypeptide having at least 85%, 90%, 95%, 97%, 98%, or 99%         sequence identity across the entire length of SEQ ID NO: 17, 19,         21, 72, 75, or 78; or (ii) a polypeptide having at one or more         conservative and/or semi-conservative amino acid substitutions         in SEQ ID NO: 17, 19, 21, 72, 75, or 78.     -   83. The method of embodiment 76, wherein the complex that binds         the promoter or 5′ UTR comprises: (i) an RNA guided DNA binding         polypeptide that is nuclease activity deficient and a guide RNA         comprising about an 18 or 19 to about a 21 or 22 nucleotide         polynucleotide sequence with is complementary to a sequence         immediately adjacent to a protospacer adjacent motif (PAM) in         the promoter or 5′ UTR; or (ii) a 20, 21, 22, 23, or 24         nucleotide polynucleotide sequence which is complementary to a         sequence immediately adjacent to a protospacer adjacent motif         (PAM) in the promoter or 5′ UTR.     -   84. The method of embodiment 83, wherein the RNA guided DNA         binding polypeptide comprises a dCAS9 polypeptide, comprises a         polypeptide having at least 85%, 90%, 95%, 97%, 98%, or 99%         sequence identity across the entire length of SEQ ID NO: 29, or         comprises a polypeptide having at one or more conservative         and/or semi-conservative amino acid substitutions in SEQ ID NO:         29.     -   85. The method of embodiment 83, wherein the RNA guided DNA         binding polypeptide comprises:         -   (i) a dCpf1 polypeptide;         -   (ii) a polypeptide having one or more conservative and/or             semi-conservative amino acid substitutions in SEQ ID NO: 44             or at least 85%, 90%, 95%, 97%, 98%, or 99% sequence             identity across the entire length of SEQ ID NO: 44 and a             D917A, E1006A, E1028A, D1255A, and/or N1257A amino acid             substitution;         -   (iii) a polypeptide having one or more conservative and/or             semi-conservative amino acid substitutions in SEQ ID NO: 45             or at least 85%, 90%, 95%, 97%, 98%, or 99% sequence             identity across the entire length of SEQ ID NO: 45 and a             D832A, E925A, and/or D1148A amino acid substitution;         -   (iv) a polypeptide having one or more conservative and/or             semi-conservative amino acid substitutions in SEQ ID NO: 46             or at least 85%, 90%, 95%, 97%, 98%, or 99% sequence             identity across the entire length of SEQ ID NO: 46 and a             D917A, E1006A, E1028A, D1255A, and/or N1257A amino acid             substitution; or         -   (v) a polypeptide having one or more conservative and/or             semi-conservative amino acid substitutions in SEQ ID NO: 47             or at least 85%, 90%, 95%, 97%, 98%, or 99% sequence             identity across the entire length of SEQ ID NO: 47 and a             D901A and/or E1228A amino acid substitution.     -   86. The method of embodiment 83, wherein the guide RNA comprises         a sequence that is complementary to either strand of an ODP2         promoter comprising SEQ ID NO: 3 or SEQ ID NO: 71 or to either         strand of an WUS2 promoter comprising SEQ ID NO: 4, wherein the         complementary sequence is immediately adjacent to a protospacer         adjacent motif (PAM) in SEQ ID NO: 3, 71, or 4.     -   87. The method of embodiment 86, wherein the guide RNA comprises         an RNA encoded by SEQ ID NO: 31, 32, 33, 34, or 35 that is         complementary to a sequence in the endogenous maize ODP2         promoter.     -   88. The method of embodiment 86, wherein the plant cell is a         maize plant cell and the guide RNA comprises an RNA encoded by         SEQ ID NO: 36, 37, 38, 39, or 40 that is complementary to a         sequence in the endogenous maize WUS2 promoter.     -   89. The method of any one of embodiments 41 to 88, wherein at         least two exogenous gene transcription agents that stimulate         transcription of the endogenous ODP2 gene and/or at least two         exogenous gene transcription agents that stimulate transcription         of the endogenous WUS2 gene are introduced into a plant cell at         step (i).     -   90. The method of embodiment 89, wherein the exogenous gene         transcription agents comprise a transcriptional activation         domain that is operably linked to or operably associated         with: (i) an artificial zinc finger (AZF) DNA binding domain         polypeptide; (ii) an artificial TALE DNA binding         polypeptide; (iii) an RNA guided DNA binding polypeptide that is         nuclease activity deficient and a guide RNA, or any combination         thereof.     -   91. The method of embodiment 89, wherein the plant cell is a         maize plant cell and the exogenous gene transcription agents         that stimulate transcription of the endogenous WUS2 gene         comprise a combination of at least two artificial TALE         transcription factors comprising a transcriptional activation         domain and an artificial TALE DNA binding polypeptide of SEQ ID         NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:72, SEQ ID NO:75,         or SEQ ID NO:78.     -   92. The method of embodiment 89, wherein the plant cell is a         maize plant cell and the exogenous gene transcription agents         that stimulate transcription of the endogenous ODP2 gene         comprise a combination of at least two distinct artificial TALE         transcription factors that each comprise a transcriptional         activation domain that is operably linked to or operably         associated with an artificial TALE DNA sequence recognition         domain polypeptide of SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27,         SEQ ID NO:81, SEQ ID NO:84, or SEQ ID NO:87.     -   93. The plant cell of any one of embodiments 1, 5, 7, 8, 9,         15-20, or 21, wherein the plant cell is a maize plant cell         comprising at least one exogenous gene transcription agent that         stimulates transcription of the endogenous WUS2 gene, wherein         expression of the endogenous WUS2 polypeptide is increased in         comparison to the expression of the endogenous WUS2 polypeptide         in a control maize plant cell, wherein the endogenous WUS2         polypeptide is encoded by an endogenous polynucleotide that is         operably linked to an endogenous maize WUS2 promoter of SEQ ID         NO:4 or an allelic variant thereof, wherein the exogenous gene         transcription agent(s) bind to DNA sequences in the endogenous         maize WUS2 promoter corresponding to residues 100 to 225 of SEQ         ID NO:4, and wherein the maize plant cell can form a regenerable         maize plant structure.     -   94. The maize plant cell of embodiment 93, wherein the exogenous         gene transcription agent(s) bind to DNA sequences in the         endogenous maize WUS2 promoter corresponding to residues 130 to         210 of SEQ ID NO:4     -   95. The maize plant cell of embodiment 93, wherein an exogenous         polynucleotide encoding a WUS2 polypeptide is absent before         and/or during the increase in expression of the endogenous WUS2         polypeptide.     -   96. The maize plant cell of embodiment 93, wherein the increase         in expression of the endogenous WUS2 polypeptide is sufficient         to increase proliferation, somatic embryogenesis, and/or         regeneration capacity of the maize plant cell in comparison to         the control plant cell.     -   97. The maize plant cell of embodiment 93, wherein the increase         in expression of the endogenous WUS2 polypeptide is sufficient         to increase transformation efficiency and/or endogenous gene         editing efficiency of the plant cell in comparison to the         control plant cell.     -   98. The maize plant cell of embodiment 93, wherein said cell is         located within or obtained from a cultured plant tissue explant,         an immature embryo, a mature embryo, a leaf, and/or callus or         optionally wherein the plant cell is located with or derived         from the L1 or L2 layer of the immature or mature embryo.     -   99. The maize plant cell of embodiment 93, wherein the         endogenous WUS2 polypeptide comprises an amino acid sequence         having at least 95%, 96%, 97%, or 99% amino acid sequence         identity across the entire length of SEQ ID NO:2.     -   100. The maize plant cell of embodiment 93, wherein the         exogenous gene transcription agent comprises: (i) a DNA binding         domain which binds to the endogenous WUS2 gene; (ii) a         transcription activation domain, wherein the transcription         activation domain is operably linked or operably associated with         the DNA binding domain; and optionally (iii) an operably linked         nuclear localization signal (NLS).     -   101. The maize plant cell of embodiment 100, wherein the DNA         binding domain that binds the WUS2 promoter comprises an         artificial zinc finger (AZF) DNA binding domain polypeptide.     -   102. The maize plant cell of embodiment 101, at least one AZF         DNA binding domain polypeptide binds to any one of SEQ ID NO:101         or 102.     -   103. The maize plant cell of embodiment 101, wherein at least         two exogenous gene transcription agents comprising an AZF DNA         binding domain polypeptide are provided and wherein one of the         AZF DNA binding domain polypeptides binds to a DNA sequence         comprising SEQ ID NO:101 and one of the AZF DNA binding domain         polypeptides binds to a DNA sequence comprising SEQ ID NO:102.     -   104. The maize plant cell of embodiment 102, wherein the AZF DNA         binding domain polypeptide comprises a polypeptide having at         least 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:105         or SEQ ID NO:106.     -   105. The maize plant cell of embodiment 101, wherein at least         two exogenous gene transcription agents comprising an AZF DNA         binding domain polypeptide are provided and wherein one of the         AZF DNA binding domain polypeptides comprises a polypeptide         having at least 95%, 98%, 99%, or 100% sequence identity to SEQ         ID NO:105 and one of the AZF DNA binding domain polypeptides         comprises a polypeptide having at least 95%, 98%, 99%, or 100%         sequence identity to SEQ ID NO:106.     -   106. The maize plant cell of embodiment 101, wherein the         exogenous gene transcription agent comprises a polypeptide         having at least 90%, 95%, 98%, 99%, or 100% sequence identity to         SEQ ID NO: 93 or 95.     -   107. The maize plant cell of embodiment 101, wherein at least         two exogenous gene transcription agents are provided and wherein         one of the exogenous gene transcription agents comprises a         polypeptide having at least 95%, 98%, 99%, or 100% sequence         identity to SEQ ID NO:93 and one of the exogenous gene         transcription agents comprises a polypeptide having at least         95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:95.     -   108. The method of any one of embodiments 41, 43-60, or 61 for         producing a regenerable plant structure, wherein the regenerable         plant structure is a regenerable maize plant structure,         comprising: (i) introducing into a maize plant cell at least one         exogenous gene transcription agent which transiently increases         expression of an endogenous WUS2 polypeptide, wherein the         expression is increased in comparison to the expression of the         endogenous WUS2 polypeptide in a control maize plant cell,         wherein the endogenous WUS2 polypeptide is encoded by an         endogenous polynucleotide that is operably linked to an         endogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic         variant thereof, and wherein the exogenous gene transcription         agent(s) bind to DNA sequences in the endogenous maize WUS2         promoter corresponding to residues 100 to 225 of SEQ ID NO:4;         and, (ii) culturing the maize plant cell to produce a         regenerable maize plant structure.     -   109. The method of embodiment 108, wherein the exogenous gene         transcription agent comprises: (i) an artificial zinc finger         (AZF) DNA binding domain which binds to of the endogenous WUS2         gene; (ii) a transcription activation domain, wherein the         transcription activation domain is operably linked or operably         associated with the domain or complex; and optionally (iii) an         operably linked nuclear localization signal (NLS).     -   110. The method of embodiment 109, wherein at least one AZF DNA         binding domain polypeptide binds to any one of SEQ ID NO:101 or         102 or wherein one AZF DNA binding domain polypeptides binds to         a DNA molecule comprising SEQ ID NO:101 and one of the AZF DNA         binding domain polypeptides binds to a DNA molecule comprising         SEQ ID NO:102.     -   111. The method of embodiment 109, wherein the AZF DNA binding         domain polypeptide comprises a polypeptide having at least 95%,         98%, 99%, or 100% sequence identity to SEQ ID NO:105 or SEQ ID         NO:106.     -   112. The method of embodiment 109, at least two exogenous gene         transcription agents comprising an AZF DNA binding domain         polypeptide are provided and wherein one of the AZF DNA binding         domain polypeptides comprises a polypeptide having at least 95%,         98%, 99%, or 100% sequence identity to SEQ ID NO:105 and one of         the AZF DNA binding domain polypeptides comprises a polypeptide         having at least 95%, 98%, 99%, or 100% sequence identity to SEQ         ID NO:106.     -   113. The method of embodiment 109, wherein the exogenous gene         transcription agent comprises a polypeptide having at least 90%,         95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 93 or 95.     -   114. The method of embodiment 109, wherein at least two         exogenous gene transcription agents are provided and wherein one         of the exogenous gene transcription agents comprises a         polypeptide having at least 95%, 98%, 99%, or 100% sequence         identity to SEQ ID NO:93 and one of the exogenous gene         transcription agents comprises a polypeptide having at least         95%, 98%, 99%, or 100% sequence identity to SEQ ID NO:95.     -   115. The method of embodiment 108, wherein the exogenous gene         transcription agent(s) bind to DNA sequences in the endogenous         maize WUS2 promoter corresponding to residues 130 to 210 of SEQ         ID NO:4.

EXAMPLES Example 1. Artificial Zinc Finger Transcription Factors (ATF) for Increasing Expression of the Endogenous Maize ODP2 and WUS2 Genes

This example provides artificial Zinc Finger Transcription Factors (ATF) for increasing expression of the endogenous maize ODP2 and WUS2 genes.

Two high quality target binding sites for an ATF (SEQ ID NO: 6 and 9) were identified in the maize ODP2 promoter region of approximately 500 bp (SEQ ID NO:3) which is proximal to the ODP2 gene transcription initiation site (e.g., mRNA cap site). A ZNF DNA binding domain (SEQ ID NO:5) and an ATF comprising that DNA binding domain (SEQ ID NO:7) were designed to bind the ZmODP2 promoter at SEQ ID NO:6. A ZNF DNA binding domain (SEQ ID NO:8) and an ATF comprising that DNA binding domain (SEQ ID NO:10) was designed to bind the ZmODP2 promoter at SEQ ID NO:9. Each of the ATFs comprise the maize opaque-2 nuclear localization signal, the artificial zinc finger DNA binding domain, and 60 amino acids from the maize C1 transcriptional activation domain.

Two high quality target binding sites for an ATF (SEQ ID NO:12 and 15) were identified in the maize WUS2 promoter region of approximately 500 bp (SEQ ID NO:4) which is proximal to the WUS2 gene transcription initiation site (e.g., mRNA cap site). A ZNF DNA binding domain (SEQ ID NO:11) and an ATF comprising that DNA binding domain (SEQ ID NO:13) was designed to bind the ZmWUS2 promoter at SEQ ID NO:12. A ZNF DNA binding domain (SEQ ID NO:14) and an ATF comprising that DNA binding domain (SEQ ID NO:16) was designed to bind the ZmWUS2 promoter at SEQ ID NO:15. Each of the ATFs comprise the maize opaque-2 nuclear localization signal, the artificial zinc finger DNA binding domain, and 60 amino acids from the maize C1 transcriptional activation domain.

Example 2. Artificial Transcription Activator-Like Effectors (aTALEs) for Increasing Expression of the Endogenous Maize ODP2 and WUS2 Genes

This example provides artificial Transcription activator-like effectors (aTALEs) for increasing expression of the endogenous maize ODP2 and WUS2 genes.

Three high quality target binding sites (SEQ ID NO: 65, 66, and 67) for three aTALEs that are spaced at intervals of about 100 nucleotides were identified in the maize WUS2 promoter region of approximately 500 bp (SEQ ID NO:4) which is proximal to the WUS2 gene transcription initiation site. A TALE DNA binding protein (SEQ ID NO:17) and an ATF comprising that DNA binding protein (SEQ ID NO:18) was designed to bind the ZmWUS2 promoter at SEQ ID NO:65. A TALE DNA binding protein (SEQ ID NO:19) and an ATF comprising that DNA binding protein (SEQ ID NO:20) was designed to bind the ZmWUS2 promoter at SEQ ID NO:66. A TALE DNA binding protein (SEQ ID NO:21) and an ATF comprising that DNA binding protein (SEQ ID NO:22) was designed to bind the ZmWUS2 promoter at SEQ ID NO:67. Each of the ATFs comprise the DNA binding protein, SV40 NLS, a 3×FLAG sequence, a VP64 transcription activation domain, and a 6×His (Histidine) domain. The aTALE ATFs targeting the WUS2 promoter can function independently or in tandem.

Three high quality target binding sites (SEQ ID NO: 74, 77, and 80) for three aTALEs were identified in the maize WUS2 promoter region of approximately 500 bp (SEQ ID NO:4) which is proximal to the WUS2 gene transcription initiation site. A TALE DNA binding protein (SEQ ID NO: 72) and an ATF comprising that DNA binding protein (SEQ ID NO: 73) was designed to bind the ZmWUS2 promoter at SEQ ID NO: 74. A TALE DNA binding protein (SEQ ID NO: 75) and an ATF comprising that DNA binding protein (SEQ ID NO: 76) was designed to bind the ZmWUS2 promoter at SEQ ID NO: 77. A TALE DNA binding protein (SEQ ID NO:78) and an ATF comprising that DNA binding protein (SEQ ID NO:79) was designed to bind the ZmWUS2 promoter at SEQ ID NO: 80. Each of the ATFs comprise a 6×His (Histidine) domain, the DNA binding protein, an SV40 NLS, and a VP64 transcription activation domain. The aTALE ATFs targeting the WUS2 promoter can function independently or in tandem.

Three high quality target binding sites (SEQ ID NO: 68, 69, and 70) for three aTALEs that are spaced at intervals of about 100 nucleotides were identified in the maize ODP2 promoter region of approximately 500 bp (SEQ ID NO: 3) which is proximal to the ODP2 gene transcription initiation codon. A TALE DNA binding protein (SEQ ID NO:23) and an ATF comprising that DNA binding protein (SEQ ID NO:24) was designed to bind the ZmODP2 promoter at SEQ ID NO:68. A TALE DNA binding protein (SEQ ID NO:25) and an ATF comprising that DNA binding protein (SEQ ID NO:26) was designed to bind the ZmODP2 promoter at SEQ ID NO:69. A TALE DNA binding protein (SEQ ID NO:27) and an ATF comprising that DNA binding protein (SEQ ID NO:28) was designed to bind the ZmODP2 promoter at SEQ ID NO:70. Each of the ATFs comprise the DNA binding domain, SV40 NLS, a 3×FLAG sequence, a VP64 transcription activation domain, and a 6×His (Histidine) domain. The three aTALE ATFs targeting the ODP2 promoter can function independently or in tandem.

Three high quality target binding sites (SEQ ID NO: 83, 86, and 89) for three aTALEs were identified in the maize ODP2 promoter region of approximately 500 bp (SEQ ID NO: 3) which is proximal to the ODP2 gene transcription initiation codon. A TALE DNA binding protein (SEQ ID NO: 81) and an ATF comprising that DNA binding protein (SEQ ID NO:82) was designed to bind the ZmODP2 promoter at SEQ ID NO: 83. A TALE DNA binding protein (SEQ ID NO: 84) and an ATF comprising that DNA binding protein (SEQ ID NO: 85) was designed to bind the ZmODP2 promoter at SEQ ID NO: 86. A TALE DNA binding protein (SEQ ID NO: 87) and an ATF comprising that DNA binding protein (SEQ ID NO: 88) was designed to bind the ZmODP2 promoter at SEQ ID NO: 89. Each of the ATFs comprise a 6×His (Histidine) domain, the DNA binding domain, SV40 NLS, and a VP64 transcription activation domain. The three aTALE ATFs targeting the ODP2 promoter can function independently or in tandem.

Example 3. Construction of dCas9 Transcription Activators and Guide RNAs Targeting ZmWUS2 and ZmODP2

This example describes an ATF comprising a nuclease deficient Cas9 DNA binding domain, guide RNAs, and vectors useful for the expression of the guide RNAs. Such ATFs and guide RNAs are designed to increase expression of the endogenous maize ODP2 and WUS2 genes.

Five crRNA (SEQ ID NO:31, 32, 33, 34, and 35) were constructed which are complementary to sequences immediately adjacent to PAM sequences in the −250 to −100 region of the maize ODP2 promoter (SEQ ID NO:3) relative to the transcription start site. Five single guide RNAs (sgRNA) incorporating the crRNA can be obtained with the sgRNA expression cassette of SEQ ID NO:41. The aforementioned sgRNAs can be expressed by substituting the aforementioned crRNA sequences for the 20 “N” (i.e., a,c,g, or t) residues in the vector of SEQ ID NO: 41 which provides for operable linkage of the crRNA sequences to a U6 promoter and sgRNA encoding sequences and introducing the vector with the substitution into a suitable host cell (e.g., a meristematic cell, a somatic cell or a reproductive cell). A dCas9 nuclease deficient RNA guided DNA binding domain (SEQ ID NO:29) is obtained and a polypeptide comprising that DNA binding domain, an SV40 NLS, a 3×FLAG sequence, a VP64 transcription activation domain, and a 6×His (Histidine) domain (SEQ ID NO:30) is designed to bind the ZmODP2 promoter when complexed with the aforementioned sgRNAs.

Five crRNA (SEQ ID NO: 36, 37, 38, 39 and 40) are constructed which are complementary to sequences immediately adjacent to PAM sequences in the −250 to −100 region of the maize WUS2 promoter (SEQ ID NO:4) relative to the transcription start site. Five single guide RNAs (sgRNA) incorporating the crRNA can be obtained with the sgRNA expression cassette of SEQ ID NO:41. The aforementioned sgRNAs can be expressed by substituting the aforementioned crRNA sequences for the 20 “N” (i.e., a,c,g, or t) residues in the vector of SEQ ID NO: 41 which provides for operable linkage of the crRNA sequences to a U6 promoter and sgRNA encoding sequences and introducing the vector with the substitution into a suitable host cell (e.g., a meristematic cell, a somatic cell or a reproductive cell). A dCas9 nuclease deficient RNA guided DNA binding domain (SEQ ID NO:29) is obtained and a polypeptide comprising that DNA binding domain, an SV40 NLS, a 3×FLAG sequence, a VP64 transcription activation domain, and a 6×His (Histidine) domain (SEQ ID NO:30) was designed to bind the ZmWUS2 promoter when complexed with the aforementioned sgRNAs.

In certain cases, the dCas9 polypeptide is expressed in E. coli, purified, and complexed in vitro with the corresponding sgRNA for delivery to a plant cell. Alternatively, the Cas9 protein and sgRNAs can be expressed from a plasmid that is delivered to the target plant cells or plant tissues.

Example 4. Use of ATFs to Obtain Regenerable Plant Structures

Developing maize embryos 8-14 days after pollination (DAP) from a variety or of a genotype that typically does not respond to biolistic or agrobacterium-mediated transformation are excised and placed on sterile plant growth media, scutellar side up. A plasmid encoding transcription activation ATFs that target the maize ODP2 and WUS2 promoter proximal regions is delivered using biolistics. The plasmid may also encode a marker gene such as GFP (or variants thereof) or mCherry fluorescent proteins to identify cells containing the plasmid.

The expected positive result is formation of regenerable plant structures comprising callus or pro-embryogenic masses from tissue that received the plasmid containing the transcription activation ATF genes after one week and no such formations on control tissue that received plasmid lacking the transcription activation ATF genes.

Example 5. Isolation of Corn Transformation Target Tissue and Biolistic Delivery of Transcription Activators in Target Tissue

Ears representing the target plant genotype are harvested approximately 8-14 DAP. The tips of developing kernels are removed with a scalpel. A fine spatula is used to gently remove the embryo from the kernel, which is then placed on callus induction media (e.g. 4 g L⁻¹ N6 salts plus N6 vitamins, 2 mg L⁻¹ 2,4-D, 2.8 g L-proline, 30 g L⁻¹ sucrose, 100 mg L⁻¹ casein hydrolysate, 100 mg L⁻¹ myo-inositol, 25 μM silver nitrate, 2.5 g L⁻¹ gelrite, pH 5.8). Embryo size is about 1.5-2.5 mm. After approximately 200 embryos are harvested, they are arranged onto four plates containing about 50 embryos each. Ideally, the scutellar surface is facing up.

The ATFs targeting ZmODP2 and ZmWUS2 (e.g., Zf ATF including SEQ ID NO:7, 10, 13, and/or 16; aTALE including SEQ ID NO:18, 20, 22, 24, 26, and/or 28; a dCas-NLS-TAD and guide RNAs including dCas9-NLS-TAD (SEQ ID NO: 30) and sgRNAs comprising crRNAs of SEQ ID NO:31, 32, 33, 34, 35, 36, 37, 38, 39, and/or 40 are encoded on plasmid DNA using suitable gene cassettes to drive their expression. The plasmid DNA is a standard high-copy E. coli vector that may or may not contain a selectable marker gene. The plasmid DNA is prepared using standard molecular biology procedures, examined for integrity and quantified. The plasmid DNA is complexed with 0.6 μm gold particles as described, for example in (Hamada et al. 2018; K. Wang and Frame 2009). The embryos are transferred to osmotic media (4 g L⁻¹ N6 salts plus N6 vitamins, 2 mg L⁻¹ 2,4-D, 0.7 g L-proline, 30 g L⁻¹ sucrose, 100 mg L⁻¹ casein hydrolysate, 100 mg L⁻¹ myo-inositol, 36.4 g sorbitol, 36.4 g mannitol, 25 μM silver nitrate, 2.5 g L⁻¹ gelrite, pH 5.8) four hours prior to bombardment. The gold particles are loaded into a BioRad PDS-1000 helium gene gun and delivered to target plant tissue following manufacturer's instructions or variations developed by other researchers. Bombarded embryos are incubated overnight in the dark at 28° C.

The following morning embryos are moved onto callus induction media as above, but with 0.8 mg L⁻¹ 2,4-D and cultured in dark at 28° C. for about 5-7 days. The tissue is examined for the formation of regenerable plant structures comprising pro-embryogenic masses starting at 5 days after bombardment. ATF efficacy is scored on the basis of pro-embryogenic mass formation compared to control tissue which received plasmid DNA lacking the ATF genes.

Example 6. Regeneration of Plants from Transformed Target Tissue

After 6-7 days on callus induction medium, the embryos are moved onto shoot formation medium (e.g. MS, 60 g L⁻¹ sucrose, 0.5 mg L⁻¹ zeatin, 0.1 mg L⁻¹ thidiazuron, 1 mg/L BAP, 0.1 mg L⁻¹ imazapyr, pH 5.8). After 2 weeks on shoot formation medium, embryos are moved to rooting medium (e.g. MS, 40 g L⁻¹ sucrose, 0.1 mg L⁻¹ imazapyr, pH 5.8) and placed under GE Ecolux® (General Electric; Boston, MA) fluorescent lights G (60 μmol m⁻² s⁻¹) with a 16-h photoperiod at 26° C. Once adequate shoots with roots form, plantlets are transferred to soil and grown to maturity in an appropriate growth environment like a greenhouse or growth chamber.

Example 7. Selection of Promoter Sequences Common Across Corn Lines for Targeting by ATFs

The promoter regions of ODP2 and WUS are sequenced and analyzed for the presence of conserved regions. These consistent sequences are desirable because the same ATF reagents can be used in diverse corn germplasm to obtain regenerable plant structures. ATF reagents can be designed to bind and activate a conserved ODP2 promoter sequence of 312 bp (SEQ ID NO:71), that is 99% identical among B73, B104, PH207, Mo17, 2FACC, LH214, LH123HT and ICI441.

Example 8. Expression of Endogenous WUS2 in Corn Protoplasts Transfected with ATFs

Corn protoplasts were transfected with vectors expressing the ATFs ZnFng-WUS1, 2, 3, and/or −4 (SEQ ID NOs: 93, 95, 97, and/or 99, respectively) or expressing GFP as a control. Endogenous RNA was extracted, and the level of WUS2 mRNA expression is quantified by RT-PCR relative to expression of the Act1 gene.

GFP, ZnFng-WUS3, or ZnFng-WUS4 expression did not significantly affect endogenous WUS2 transcription over background levels in mock transfected protoplasts. In contrast, ZnFng-WUS1 and ZnFng-WUS2 increased WUS2 transcription over the background levels in mock transfected protoplasts. ZnFng-WUS1 promotes WUS2 expression increase to about 10% relative to actin and normalized by transfection efficiency (in the 8-12% range in different experiments). ZnFng-WUS2 promoted a WUS2 expression increase of about 50% relative to actin and normalized by transfection efficiency (in the 15 to 60% range in different experiments). Mock transfected protoplasts had a nearly zero WUS2 expression level relative to actin.

Example 9. Biological Sequences

This example provides non-limiting embodiments of proteins, promoters, and coding sequences referred to herein. Biological sequences and their SEQ ID NOs are set forth in Table 1.

TABLE 1 Biological Sequences SEQ ID NO: DESCRIPTION SEQUENCE COMMENTS 1 Endogenous MATVNNWLAFSLSPQELPPSQTTDS Maize TLISAATADHVSGDVCFNIPQDWSM ODP2 RGSELSALVAEPKLEDFLGGISFSE polypeptide QHHKSNCNLIPSTSSTVCYASSAAS TGYHHQLYQPTSSALHFADSVMVAS SAGVHDGGSMLSAAAANGVAGAASA NGGGIGLSMIKNWLRSQPAPMQPRA AAAEGAQGLSLSMNMAGTTQGAAGM PLLAGERARAPESVSTSAQGGAVVV TAPKEDSGGSGVAGALVAVSTDTGG SGGASADNTARKTVDTFGQRTSIYR GVTRHRWTGRYEAHLWDNSCRREGQ TRKGRQVYLGGYDKEEKAARAYDLA ALKYWGATTTTNFPVSNYEKELEDM KHMTRQEFVASLRRKSSGFSRGASI YRGVTRHHQHGRWQARIGRVAGNKD LYLGTFSTQEEAAEAYDIAAIKFRG LNAVTNFDMSRYDVKSILDSSALPI GSAAKRLKEAEAAASAQHHHAGVVS YDVGRIASQLGDGGALAAAYGAHYH GAAWPTIAFQPGAATTGLYHPYAQQ PMRGGGWCKQEQDHAVIAAAHSLQD LHHLNLGAAGAHDFFSAGQQAAAAA AMHGLASIDSASLEHSTGSNSVVYN GGVGDSNGASAVGSGGGYMMPMSAA GATTTSAMVSHEQMHARAYDEAKQA AQMGYESYLVNAENNGGGRMSAWGT VVSAAAAAAASSNDNIAADVGHGGA QLFSVWNDT 2 Endogenous MAANAGGGGAGGGSGSGSVAAPAVC Maize RPSGSRWTPTPEQIRMLKELYYGCG WUS2 IRSPSSEQIQRITAMLRQHGKIEGK polypeptide NVFYWFQNHKARERQKRRLTSLDVN VPAAGAADATTSQLGVLSLSSPPSG AAPPSPTLGFYAAGNGGGSAGLLDT SSDWGSSGAAMATETCFLQDYMGVT DTGSSSQWPCFSSSDTIMAAAAAAA RVATTRAPETLPLFPTCGDDDDDDS QPPPRPRHAVPVPAGETIRGGGGSS SSYLPFWGAGAASTTAGATSSVAIQ QQHQLQEQYSFYSNSTQLAGTGSQD VSASAAALELSLSSWCSPYPAAGSM 3 Endogenous AAATGGCCGTGACAACGTATACTAT 500 bp promoter Maize TATCGAGTAAAAGGTCGCCACTTTA sequence; TATA ODP2 GTAGTACATGTACATGCATGCGCAG box underlined; promoter ATACATCATCAGGTACTCATATATG 5′-UTR in lower and GGCACACATATAGACATGTTTTGAG case italics 5′-UTR GAAAATGAGACAAAGTATAGTGGAG ACTTCCCTAGAAAGCAGAAGAAAAA GAAGTGGTTTATGTTCCGTTAAATC ATACTACAACTTTTTTTTATTATAC TCTCCATTTTGTCATCATTAGGTAC TCATATATGGGCACACATATAGTAC TGCCAATTTTTCTTGCTAAAAAAAG TTCCACTATATATATGTATGTATGC ACAAATAAACTAATTTTCTTAGAAA AGAAAACCGGTGTAATACATACTAA GGGCTAGTTTGGGAACCCTGGTTTT CTAAGGAATTTTATTTTTCCAAAAA AAATAGTTTATTTTTCCTTCGGAAA TTAGGAATCTCTTATA AAATTCGAGTTCCCAAACTATTC CTAATATATATatcatactctccat cagtctatatatagattacatatag taagtatagagtatctcgctatcac atagtgccactaatcttctggagtg taccagttgtataaatatctatcag tatcagcactactgtttgctgaata ccccaaaactctctgcttgacttct cttccctaacctttgcactgtccaa aatggcttcctgatcccctcacttc ctcgaatcattctaagaagaaactc aagccgctaccattaggggcagatt aattgctgcactttcagataatcta cc 4 Endogenous CATTGAACAATGGAGCTGCAAGAGCAATGATG 609 bp WUS2 Maize CACTAGCTAGTGTAATGCAGTGCATGCATGGT promoter and WUS2 AGATTGGTAGCTAGCCTTTGCAGTTTGCACCA 5′UTR sequence: promoter GGCACCAGCAGCAGCTAGAAGACGACAGACG TATA box and ACAGGGGCTT

CAG underlined; 5′-UTR TTGCCAGTTGCCACAAGGGGAGCCTG

binding sites for

ATGATAGCTCTGTCTCTCTCA ZnFng-WUS1 CACACACACACAGTCACACAGAGACACGCAAA AZFDNA TGACTTCTGTCTCTAACTCTTCCAAATTTCGAA BINDING SITE GCGGCCAATGCAAGAGCCAGCCCCCGGCCGT (SEQ ID NO: ATGTCAACTTCACTTGTCTCTCTCCAAAAGATA 101) at TCGTATCACCCATGGGCAATGGCCATGACCCC nucleotides 138- CCTCCCAGCCCCAACCTATATCACCTAGCGCAG 155 and ZnFng- CTACGCTCTCTTCTCCCGCTCTCGCTCTCTGCTG WUS2 AZF DNA GCTGCATGCTAGCTACCTTCTAGCTATCTAGCC BINDING SITE TCTAGCTCCAATGCACTCCCTCCTTATAAACAA (Reverse GGAACCCTCCTTCGGCTCTCTTGCCATAGACCG complement of GACACCGGAGAGCTAGGTCACAGAAGCGCTC SEQ ID NO: 102) AGGAAGGCCGCTGCGCTGAGATAGAGGC at nucleotides 185-202 to double underlined and in bold 5 Zinc Finger DNA LEPGEKPYKCPECGKSFSQSSSLVRHQR TGEKP linkers binding domain THTGEKPYKCPECGKSFSRADNLTEHQ separating zinc targeting ZmODP2 RTHTGEKPYKCPECGKSFSTSGNLTEHQ fingers are gene sequence at RTHTGEKPYKCPECGKSFSTSGNLTEHQ underlined position 73 RTHTGEKPYKCPECGKSFSQKSSLIAHQ RTHTGEKPYKCPECGKSFSRADNLTEH QRTHTGKKTS 6 ZmODP2 gene 5′-cagatacatcatcaggta-3′ sequence at position 73 7 Complete Zf ATF MrkrkesnresarrsrrsrvrkkvLEPGEKPYKCPE maize opaque-2 targeting ZmODP2 CGKSFSQSSSLVRHQRTHTGEKPYKCPE nuclear at position 73 CGKSFSRADNLTEHQRTHTGEKPYKCP localization signal ECGKSFSTSGNLTEHQRTHTGEKPYKCP is lower case and ECGKSFSTSGNLTEHQRTHTGEKPYKCP underlined; maize ECGKSFSQKSSLIAHQRTHTGEKPYKCP C1 transcription ECGKSFSRADNLTEHQRTHTGKKTSAG activation domain SSDDCSSAASVSLRVGSHDEPCFSGDGD double underlined GDWMDDVRALASFLESDEDWIRCQTA GQLA 8 Zinc Finger DNA LEPGEKPYKCPECGKSFSQSGDLRRHQ binding domain RTHTGEKPYKCPECGKSFSTSGNLTEHQ targeting ZmODP2 RTHTGEKPYKCPECGKSFSQSSSLVRHQ gene sequence at RTHTGEKPYKCPECGKSFSTSGNLTEHQ position 51 RTHTGEKPYKCPECGKSFSQSSSLVRHQ RTHTGEKPYKCPECGKSFSQSSSLVRHQ RTHTGKKTS 9 ZniODP2 gene 5′-gtagtacatgtacatgca-3′ sequence at position 51 10 Complete Zf ATF Mrkrkesnresarrsrrsrvrkkv maize opaque-2 targeting ZmODP2 LEPGEKPYKCPE nuclear at position 51 CGKSFSQSGDLRRHQRTHTGEKPYKCP localization signal ECGKSFSTSGNLTEHQRTHTGEKPYKCP is lower case and ECGKSFSQSSSLVRHQRTHTGEKPYKCP underlined; maize ECGKSFSTSGNLTEHQRTHTGEKPYKCP C1 transcription ECGKSFSQSSSLVRHQRTHTGEKPYKCP ECGKSFSQSSSLVRHQRTHTGKKTSAGS activation domain SDDCSSAASVSLRVGSHDEPCFSGDGD double underlined GDWMDDVRALASFLFSDFDWLRCQTA GQLA 11 Zinc Finger DNA LEPGEKPYKCPECGKSFSQAGHLASHQ binding domain RTHTGEKPYKCPECGKSFSTSGNLTEHQ targeting RTHTGEKPYKCPECGKSFSRSDKLTEHQ ZmWUS2 gene RTHTGEKPYKCPECGKSFSRNDALTEH sequence at QRTHTGEKPYKCPECGKSFSQSSNLVR position 83 HQRTHTGEKPYKCPECGKSFSTTGNLT VHQRTHTGKKTS 12 ZmWUS2 gene 5′-AATGAACTGCGGCATTGA-3′ sequence at position 83 13 Complete Zf ATF MrkrkesnresarrsrrsrvrkkvLEPGEKPYKCPE maize opaque-2 targeting CGKSFSQAGHLASHQRTHTGEKPYKCP nuclear ZmWUS2 at ECGKSFSTSGNLTEHQRTHTGEKPYKCP localization signal position 83 ECGKSFSRSDKLTEHQRTHTGEKPYKCP is lower case and ECGKSFSRNDALTEHQRTHTGEKPYKC underlined; maize PECGKSFSQSSNLVRHQRTHTGEKPYK C1 transcription CPECGKSFSTTGNLTVHQRTHTGKKTS activation domain AGSSDDCSSAASVSLRVGSHDEPCFSGD double underlined GDGDWMDDVRALASFLFSDEDWLRCQ TAGQLA 14 Zinc Finger DNA LEPGEKPYKCPECGKSFSRSDNLVRHQ binding domain RTHTGEKPYKCPECGKSFSRRDELNVH targeting QRTHTGEKPYKCPECGKSFSSPADLTRH ZmWUS2 gene QRTHTGEKPYKCPECGKSFSQAGHLAS sequence at HQRTHTGEKPYKCPECGKSFSTSGNLTE position 92 HQRTHTGEKPYKCPECGKSFSRSDKLTE HQRTHTGKKTS 15 ZmWUS2 gene 5′-CGGCATTGAACAATGGAG-3′ sequence at position 92 16 Complete Zf ATF MrkrkesnresarrsrrsrvrkkvLEPGEKPYKCPE maize opaque-2 targeting CGKSFSRSDNLVRHQRTHTGEKPYKCP nuclear ZmWUS2 at ECGKSFSRRDELNVHQRTHTGEKPYKC localization signal position 92 PECGKSFSSPADLTRHQRTHTGEKPYKC is lower case and PECGKSFSQAGHLASHQRTHTGEKPYK underlined; maize CPECGKSFSTSGNLTEHQRTHTGEKPYK C1 transcription CPECGKSFSRSDKLTEHQRTHTGKKTSA activation domain GSSDDCSSAASYSLRYGSHDEPCFSGDG double underlined DGDWMDFWRALASFLESDEDWIRCQT AGQLA 17 ZmWUS2-TALE- Mapkkkrkvdykdhdgdykdhdidykdd N-terminus in 1 N-terminus, Ddkgtvdlrtlgysqqqqekikpkvrst lower case; RVD RVD, and C- Vaqhhealvghgfthahivalsqhpaal domain in in temiinal domain Gtvavkyqdmiaalpeatheaivgvgkq uppercase with Wsgaralealltvagelrgpplqldtgq hypervariable Llkiakrggvtaveavhawrnaltgapl diresidues double Nltpeqvvai underlined; C- LTPEQVVAIASNHGGKQ terminus in ALETVQRLLPVLCQAHGLTPEQVVAIAS lowercase italics; NHGGKQALETVQRLLPVLCQAHGLTPE Binds upstream of QVVAIASHDGGKQALETVQRLLPVLCQ the ZmWUS2 AHGLTPEQVVAIASHDGGKQALETVQR transcription start LLPVLCQAHGLTPEQVVAIASHDGGKQ site ALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQAHLTPEQ VVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNHGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASMGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRL LPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASN HGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNIGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIAS NHGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASHDGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNGGG sivaqlsrpdpalaaltndhlvalaclgg rpaldavkkglphapalikrtnrripert shrva 18 ZmWUS2-TALE- mapkkkrkvdykdhdgdykdhdidy ZmWUS2-TALE- 1 Artificial kddddkgtvdlrtlgysqqqqekik 1 N terminus, Transcription pkvrstvaqhhealvghgfthahiv RVD domain, C- Factor alsqhpaalgtvavkyqdmiaalpe terminus in lower atheaivgvgkqwsgaralealltv case; SV40 agelrgpplqldtgqllkiakrggv Nuclear taveavhawmaltgaplnltpeqvv Localization ailtpeqvvaiasnhggkqaletvq Sequence in rllpvlcqahgltpeqvvaiasnhg uppercase, gkqaletvqrllpvlcqahgltpeq underlined; waiashdggkqaletvqrllpvlcq 3xFLAG ahgltpeqvvaiashdggkqaletv sequence qrllpvlcqahgltpeqvvaiashdg lowercase, gkqaletvqrllpvlcqahgltpeq underlined; VP64 vvaiasngggkqaletvqrllpvlc transcriptional qahltpeqvvaiashdggkqaletv activation qrllpvlcqahgltpeqvvaiasngg domain gkqaletvqrllpvlcqahgltpeq is lowercase, vvaiasnhggkqaletvqrllpvlc double qahgltpeqvvaiashdggkqaletv underlined, qrllpvlcqahgltpeqvvaiasni italics; ggkqaletvqrllpvlcqahgltpe 6xHis tag qvvaiasngggkqaletvqrllpvl at C-terminus cqahgltpeqvvaiashdggkqale tvqrllpvlcqahgltpeqvvaias hdggkqaletvqrllpvlcqahglt peqvvaiasngggkqaletvqrllp vlcqahgltpeqvvaiashdggkqa letvqrllpvlcqahgltpeqvvai ashdggkqaletvqrllpvlcqahg ltpeqvvaiasngggkqaletvqrl lpvlcqahgltpeqvvaiashdggk qaletvqrllpvlcqahgltpeqvv aiasniggkqaletvqrllpvlcqa hgltpeqvvaiasngggkqaletvqr llpvlcqahgltpeqvvaiasnhgg kqaletvqrllpvlcqahgltpeqv vaiasniggkqaletvqrllpvlcq ahgltpeqvvaiasngggkqaletv qrllpvlcqahgltpeqvvaiasni ggkqaletvqrllpvlcqahgltpe qvvaiasnhggkqaletvqrllpvl cqahgltpeqvvaiashdggkqalet vqrllpvlcqahgltpeqvvaiasn gggsivaqlsrpdpalaaltndhlv alaclggrpaldavkkglphapali krtnrripertshrvaPKKKRKVSS dykdhdgdykdhdidykddddkAAG GGGSGRA

HHHHHH 19 ZmWUS2-TALE- Mapkkkrkvdykdhdgdykdhdidyk N-terminus in 2 N-terminus, Ddddkgtvdlrtlgysqqqqekikpk lower case; RVD RVD, and C- vrstvaqhhealvghgfthahivals domain in in terminal qhpaalgtvavkyqdmiaalpeath uppercase with domain eaivgvgkqwsgaraleal1tvagel hypervariable rgpplqldtgqllkiakrggvtavea diresidues vhawmaltgaplnltpeavvaiLTPE underlined; C- QVVAIASHDGGKQA terminus in LETVQRLLPVLCQAHGLTPEQVVAIAS lowercase italics; NGGGKQALETVQRLLPVLCQAHGLTPE Binds upstream of QVVAIASNGGGKQALETVQRLLPVLCQ the ZmWUS2 AHGLTPEQVVAIASHDGGKQALETVQR transcription LLPVLCQAHGLTPEQVVAIASHDGGKQ start ALETVQRLLPVLCQAHGLTPEQVVAIAS site NIGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQ ahgltpeqvvaiasniggkqaletvqr LLPVLCQAHGLTPEQVVAIASNGGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNHGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNHGGKQALETVQR LLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIAS NHGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNHGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNHGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNIGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNGGG sivaqlsrpdpalaaltndhlvalac lggrpaldavkkglphapalikrtnm pertshrva 20 ZmWUS2-TALE- mapkkkrkvdykdhdgdykdhdidy ZmWUS2-TALE- 2 Artificial kddddkgtvdlrtlgysqqqqeki 2 N terminus, Transcription kpkvrstvaqhhealvghgfthahi RVD domain, C- Factor valsqhpaalgtvavkyqdmiaalp terminus in lower eatheaivgvgkqwsgaraleallt case; SV40 vagelrgpplqldtgqllkiakrgg Nuclear vtaveavhawrnaltgaplnltpeq Localization vvailtpeqvvaiashdggkqalet Sequence in vqrllpvlcqahgltpeqvvaiasn uppercase, double gggkqaletvqrllpvlcqahgltp underlined; eqvvaiasngggkqaletvqrllpv 3xFLAG lcqahgltpeqvvaiashdggkqal sequence etvqrllpvlcqahgltpeqvvaia lowercase, shdggkqaletvqrllpvlcqahgl underlined; VP64 tpeqvvaiasniggkqaletvqrll transcriptional pvlcqahgltpeqvvaiasniggkq activation domain aletvqrllpvlcqahgltpeqvva is lowercase, iasniggkqaletvqrllpvlcqah double gltpeqvvaiasngggkqaletvqr underlined, llpvlcqahgltpeqvvaiasngggk italics; 6xHis tag qaletvqrllpvlcqahgltpeqvv at C-terminus aiashdggkqaletvqrlIpvlcqa hgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiasnhgg kqaletvqrllpvlcqahgltpeqv vaiasniggkqaletvqrllpvlcq ahgltpeqvvaiasniggkqaletv qrllpvlcqahgltpeqvvaiasnh ggkqaletvqrlIpvlcqahgltpe qvvaiashdggkqaletvqrllpvl cqahgltpeqvvaiasnhggkqale tvqrllpvlcqahgltpeqvvaias nhggkqaletvqrllpvlcqahglt peqvvaiashdggkqaletvqrllp vlcqahgltpeqvvaiashdggkqa letvqrllpvlcqahgltpeqvvai asniggkqaletvqrllpvlcqahg ltpeqvvaiasniggkqaletvqrl lpvlcqahgltpeqvvaiasngggk qaletvqrllpvlcqahgltpeqvv aiasnhggkqaletvqrllpvlcqa hgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiasnig gkqaletvqrllpvlcqahgltpeq vvaiasniggkqaletvqrllpvlc qahgltpeqvvaiasngggsivaql srpdpalaaltndhlvalaclggrp aldavkkglphapalikrtnrripe rtshrva

Sdykdhdgdy kdhdidykddddkAAGGGGSGRA

HHHHHH 21 Zm WUS2-T ALE- Mapkkkrkvdykdhdgdykdhdidyk N-terminus in 3 N-termin ddddkgtvdlrtlgysqqqqekikpkv lower case; RVD us, rstvaqhhealvghgfthahivals domain in RVD, and C- qhpaalgtvavkyqdmiaalpeath uppercase with terminal eaivgvgkqwsgaralealltvage hypervariable domain lrgpplqldtgqllkiakrggvtav di residues eavhawrnaltgaplnltpeqvvai underlined; C- LTPEQVVAIASNIG terminus in GKQALETVQRLLPVLCQAHGLTPEQ lowercase VVAIASNGGGKQALETVQRLLPVLC italics; QAHGLTPEQVVAIASHD Binds GGKQALETVQRLLPVLCQAHGL upstream of TPEQVVAIASNHGG the ZmWUS2 KQALETVQRLLPVLCQAHGLTPEQV transcription VAIASNGGGKQALETVQRLLPVLCQ start AHGLTPEQVVAIASNJGGKQALETV site QRLLPVLCQAHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASNIGGKQALETVQ RLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLLPV LCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAH GLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASNGGGKQALETVQRLLPVLCQAHG LTPEQVVAIASNHG GKQALETVQRLLPVLCQAHGLTPEQ VVAIASNHGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIA SHDGGKQALETVQR LLPVLCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVL CQAHGLTPEQVVAIASNGGGKQALE TVQRLLPVLCQAHGLTPEQVVAIAS NHGGKQALETVQRL LPVLCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGL TPEQVVAIASHDGG KQALETVQRLLPVLCQAHGLTPEQV VAIASHDGGKQALE TVQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLT PEQVVAIASNGGG sivaqlsrpdpalaaltndhlvala clggrpaldavkkglphapalikrt nrripertshrva 22 ZmWUS2-TALE- mapkkkrkvdykdhdgdykdhdidy ZmWUS2-TALE- 3 Artificial kddddkgtvdlrtlgysqqqqekik 3 N terminus, Transcription pkvrstvaqhhealvghgfthahiv RVD domain, C- Factor alsqhpaalgtvavkyqdmiaalpe Terminus atheaivgvgkqwsgaralealltv in lower agelrgpplqldtgqllkiakrggv case; SV40 taveavhawrnaltgaplnltpeqv Nuclear vailtpeqvvaiasniggkqaletv Localization qrllpvlcqahgltpeqvvaiasng Sequence in ggkqaletvqrllpvlcqahgltpe uppercase, qvvaiashdggkqaletvqrllpvl underlined; cqahgltpeqvvaiasnhggkqale 3xFLAG tvqrllpvlcqahgltpeqvvaias sequence ngggkqaletvqrllpvlcqahglt lowercase, peqvvaiasniggkqaletvqrllp underlined; vlcqahgltpeqvvaiasngggkqa VP64 letvqrllpvlcqahgltpeqvvai transcriptional ashdggkqaletvqrllpvlcqahg activation ltpeqvvaiasniggkqaletvqrl domain lpvlcqahgltpeqvvaiashdggk is lowercase, qaletvqrllpvlcqahgltpeqvv double aiashdggkqaletvqrllpvlcqa hgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiasnig gkqaletvqrllpvlcqahgltpeq vvaiasngggkqaletvqrllpvlc qahgltpeqvvaiasnhggkqalet vqrllpvlcqahgltpeqvvaiasn hggkqaletvqrllpvlcqahgltp eqvvaiashdggkqaletvqrllpv lcqahg ltpeqvvaiasniggkqaletvqrl underlined, lpvlcqahgltpeqvvaiasngggk italics; qaletvqrllpvlcqahgltpeqvv 6xHis tag aiasnhggkqaletvqrllpvlcqa at C-terminus hgltpeqvvaiasniggkqaletvq rllpvlcqahgltpeqvvaiashdg gkqaletvqrllpvlcqahgltpeq vvaiashdggkqaletvqrllpvlc qahgltpeqvvaiashdggkqalet vqrllpvlcqahgltpeqvvaiash dggkqaletvqrllpvlcqahgltp eqvvaiashdggkqaletvqrllpv lcqahgltpeqvvaiashdggkqal etvqrllpvlcqahgltpeqvvaia sngggsivaqlsrpdpalaaltndh lvalaclggrpaldavkkglphapa likrtnrripertshrvaPKKKRKV SSdykdhdydykdhdidykddddkA AGGGGSGRA

HHHHHH 23 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N-terminus in 1 N-terminus, kddddkgtvdlrtlgysqqqqekik lower case; RVD RVD, and C- pkvrstvaqhhealvghgfthahiv domain in terminal domain alsqhpaalgtvavkyqdmiaalpe uppercase with atheaivgvgkqwsgaralealltv hypervariable agelrgpplqldtgqllkiakrggv di residues taveavhawrnaltgaplnltpeqv underlined; C- vaiLTPEQVVAIASNI terminus in GGKQALETVQRLLPVLCQAHGLT lowercase PEQVVAIASNGGGK italics; QALETVQRLLPVLCQAHGLTPEQVV Binds AIASNGGGKQALET upstream of VQRLLPVLCQAHGLTPEQVVAIAS the ZmODP2 HDGGKQALETVQRLL transcription PVLCQAHGLTPEQVVAIAS start HDGGKQALETVQRLLPVLCQ site AHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAHGLT PEQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLL PVLCQAHGLTPEQVVAIA NI GGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAHGLT PEQVVAIASMGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAH GLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASNIGGKQALETVQ RLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPV LCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAH GLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQA LETVQRLLPV LCQAHGLTPEQVVAIAS NIGGKQALETVQRLLPVLCQAH GLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPV LCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAH GLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASNGGGsivaqlsv rpdpalaaltndhlvalaclggrpa ldavkkglphapalikrfnrriper tshrva 24 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidyk Zm ODP2-TALE- 1 Artificial ddddkgtvdlrtlgysqqqqekikp 1 N terminus, Transcription kvrstvaqhhealvghgfthahiva RVD domain, C- Factor lsqhpaalgtvavkyqdmiaalpea terminus theaivgvgkqwsgaralealltva in lower gelrgpplqldtgqllkiakrggvt case; SV40 aveavhawrnaltgaplnltpeqvv Nuclear ailtpeqvvaiasniggkqaletvq Localization rllpvlcqahgltpeqvvaiasngg Sequence in gkqaletvqrllpvlcqahgltpeq uppercase, vvaiasngggkqaletvqrllpvlc underlined; qahgltpeqvvaiashdggkqalet 3xFLAG vqrllpvlcqahgltpeqvvaiash sequence dggkqaletvqrllpvlcqahgltp lowercase, eqvvaiasngggkqaletvqrllpv underlined; lcqahgltpeqvvaiasniggkqal VP64 etvqrllpvlcqahgltpeqvvaia transcriptional sniggkqaletvqrllpvlcqahgl activation tpeqvvaiasngggkqaletvqrll domain pvlcqahgltpeqvvaiasniggkq is lowercase, aletvqrllpvlcqahgltpeqvva double iasngggkqaletvqrllpvlcqah underlined, gltpeqvvaiasniggkqaletvqr italics; llpvlcqahgltpeqvvaiasnggg 6xHis tag kqaletvqrllpvlcqahgltpeqv at C-terminus vaiasniggkqaletvqrllpvlcq ahgltpeqvvaiasngggkqaletv qrllpvlcqahgltpeqvvaiasni ggkqaletvqrllpvlcqahgltpe qvvaiasngggkqaletvqrllpvl cqahgltpeqvvaiashdggkqale tvqrllpvlcqahgltpeqvvaias niggkqaletvqrllpvlcqahglt peqvvaiasngggkqaletvqrllp vlcqahgltpeqvvaiasniggkqa letvqrllpvlcqahgltpeqvvai ashdggkqaletvqrllpvlcqahg ltpeqvvaiasngggkqaletvqrl lpvlcqahgltpeqvvaiashdggk qaletvqrllpvlcqahgltpeqvv aiasngggkqaletvqrllpvlcqa hgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiashdg gkqaletvqrllpvlcqahgltpeq vvaiasniggkqaletvqrllpvlc qahgltpeqvvaiasngggsivaql srpdpalaaltndhlvalaclggrp aldavkkglphapalikrtnrripe rtshrvaPKKKRKVSS dykdhdgdykdhdidykddddk AAGGGGSGRAd

HHHHHH 25 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N-terminus in 2 N-terminus, kddddkgtvdlrtlgysqqqqekik lower case; RVD RVD, and C- pkvrstvaqhhealvghgfthahiv domain in terminal alsqhpaalgtvavkyqdmiaalpe uppercase with domain atheaivgvgkqwsgaralealltv hypervariable agelrgpplqldtgqllkiakrggv di residues taveavhawrnaltgaplnltpeqv underlined; C- vaiLTPEQVVAIASHD terminus in GGKQALETVQRLLPVLCQAHGLT lowercase italics; PEQVVAIASNGGGK Binds upstream of QALETVQRLLPVLCQAHGLTPEQVV the ZmODP2 AIASNGGGKQALET transcription start VQRLLPVLCQAHGLTPEQVVAIAS site HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLT PEQVVAIASNHGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS NHGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLT PEQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQ AHGLTPEQVVAIASMGGKQALETVQ RLLPVLCQAHGLTPEQVVAIAS NHGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNG GGKQALETVQRLLPVLCQAH GLTPEQVVAIASNG GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNHGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASNGGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASN1G GKQALETVQRLLPVLCQAHGLTPEQ VVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIA SNIGGKQALETVQR LLpvlcqahgltpeqvvaiasmggk qaleTVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTP EQVVAIASNGGG sivaqlsrpdpalaaltndhlv alaclggrpaldavkkglphap alikrtnrripertshrva 26 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidykdd N terminus, RVD 2 Artificial ddkgtvdlrtlgysqqqqekikpkvr domain, C- Transcription stvaqhhealvghgfthahivalsq terminus Factor hpaalgtvavkyqdmiaalpeathe in lower aivgvgkqwsgaralealltvagel case; SV40 rgpplqldtgqllkiakrggvtave Nuclear avhawrnaltgaplnltpeqvvailt Localization peqvvaiashdggkqaletvqrllp Sequence in vlcqahgltpeqvvaiasngggkqa uppercase, letvqrllpvlcqahgltpeqvvai underlined; asngggkqaletvqrllpvlcqahg 3xFLAG ltpeqvvaiashdggkqaletvqrl sequence lpvlcqahgltpeqvvaiasngggk lowercase, qaletvqrllpvlcqahgltpeqvv underlined; aiasnhggkqaletvqrllpvlcqa VP64 hgltpeqvvaiasnhggkqaletvq transcriptional rllpvlcqahgltpeqvvaiasnig activation gkqaletvqrllpvlcqahgltpeq domain vvaiasnhggkqaletvqrllpvlc is lowercase, qahgltpeqvvaiasngggkqalet double vqrllpvlcqahgltpeqvvaiasn underlined, hggkqaletvqrllpvlcqahgltp italics; eqvvaiasngggkqaletvqrllpv 6xHis tag lcqahgltpeqvvaiasniggkqal at C-terminus etvqrllpvlcqahgltpeqvvaia shdggkqaletvqrllpvlcqahgl tpeqvvaiashdggkqaletvqrll pvlcqahgltpeqvvaiasniggkq aletvqrllpvlcqahgltpeqvva iasnhggkqaletvqrllpvlcqah gltpeqvvaiasngggkqaletvqr llpvlcqahgltpeqvvaiasngggk qaletvqrllpvlcqahgltpeqvv aiasnhggkqaletvqrllpvlcqa hgltpeqvvaiasngggkqaletvq rllpvlcqahgltpeqvvaiasnig gkqaletvqrllpvlcqahgltpeq vvaiasngggkqaletvqrllpvlc qahgltpeqvvaiasniggkqalet vqrllpvlcqahgltpeqvvaiasn iggkqaletvqrllpvlcqahgltp eqvvaiasniggkqaletvqrllpv lcqahgltpeqvvaiasngggkqal etvqrllpvlcqahgltpeqvvaia sniggkqaletvqrllpvlcqahgl tpeqvvaiasngggsivaqlsrpdp alaaltndhlvalaclggrpaldav kkalgphaalikrtnrripertshrv a

SS dykdhdgdykdhdidykddddk AAGGGGSGRA

HHHHHH 27 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N-terminus in 3 N-terminus, kddddkgtvdlrtlgysqqqqekik lower case; RVD RVD, and C- pkvrstvaqhhealvghgfthahiv domain in terminal alsqhpaalgtvavkyqdmiaalpe uppercase with domain atheaivgvgkqwsgaralealltv hypervariable agelrgpplqldtgqllkiakrggv diresidues taveavhawrnaltgaplnltpeqv underlined; C- vaiLTPEQVVAIASNHG terminus in GKQALETVQRLLPVLCQAHGLT lowercase PEQVVAIASHDGGKQA italics; LETVQRLLPVLCQAHGLTPEQVV Binds AIASNIGGKQALETVQ upstream of RLLPVLCQAHGLTPEQVVAIAS the ZmODP2 HDGGKQALETVQRLL transcription PVLCQAHGLTPEQVVAIASNG start GGKQALETVQRLLPVLCQ site AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLT PEQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNI GGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLT PEQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNH GGKQALETVQRLLPVLCQAHGLT PEQVVAIASHDGGK QALETVQRLLPVLCQAHGLTPEQVV AIASNGGGKQALET VQRLLPVLCQAHGLTPEQVVAIAS NGGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQ AHGLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHG PLTEQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPE QVVAIASNHGGKQALET VQRLLPVLCQAHGLTPEQVVAIASM GGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQA LETVQRLLPVLCQAHGLTPEQVVAI ASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIAS HDGGKQALETVQRLLPV LCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAH GLTPEQVVAIASHD GGKQALETVQRLLPVLCQAHGLTP EQVVAIASNGGG sivaqlsrpdpalaaltndhlv alaclggrpaldavkkglphap alikrtnrripertshrva 28 Zm ODP2-TALE- mapkkkrkvdykdhdgdykdhdidy N terminus, RVD 3 Artificial kddddkgtvdlrtlgysqqqqekik domain, C- TranscriptiQn pkvrstvaqhhealvghgfthahiv terminus in lower FactQr alsqhpaalgtvavkyqdmiaalpe case; SV40 atheaivgvgkqwsgaralealltv Nuclear agelrgpplqldtgqllkiakrggv Localization taveavhawrnaltgaplnltpeqv Sequence in vailtpeqvvaiasnhggkqaletv uppercase, qrllpvlcqahgltpeqvvaiashd underlined; ggkqaletvqrllpvlcqahgltpe 3xFLAG qvvaiasniggkqaletvqrllpvl sequence cqahgltpeqvvaiashdggkqale lowercase, tvqrllpvlcqahgltpeqvvaias underlined; VP64 ngggkqaletvqrllpvlcqahglt transcriptional peqvvaiasnhggkqaletvqrllp activation domain vlcqahgltpeqvvaiasngggkqa is lowercase, letvqrllpvlcqahgltpeqvvai double ashdggkqaletvqrllpvlcqahg underlined, ltpeqvvaiashdggkqaletvqrl italics; 6xHis tag lpvlcqahgltpeqvvaiasniggk at C-terminus qaletvqrllpvlcqahgltpeqvv aiasniggkqaletvqrllpvlcqa hgltpeqvvaiasniggkqaletvq rllpvlcqahgltpeqvvaiasnig gkqaletvqrllpvlcqahgltpeq vvaiasngggkqaletvqrllpvlc qahgltpeqvvaiasnhggkqalet vqrllpvlcqahgltpeqvvaiasn hggkqaletvqrllpvlcqahgltp eqvvaiashdggkqaletvqrllpv lcqahgltpeqvvaiasngggkqal etvqrllpvlcqahgltpeqvvaia sngggkqaletvqrllpvlcqahgl tpeqvvaiashdggkqaletvqrll pvlcqahgltpeqvvaiashdggkq aletvqrllpvlcqahgltpeqvva iasngggkqaletvqrllpvlcqah gltpeqvvaiasnhggkqaletvqr llpvlcqahgltpeqvvaiasnigg kqaletvqrllpvlcqahgltpeqv vaiasngggkqaletvqrllpvlcq ahgltpeqvvaiashdggkqaletv qrllpvlcqahgltpeqvvaiashd ggkqaletvqrllpvlcqahgltpe qvvaiashdggkqaletvqrllpvl cqahgltpeqvvaiashdggkqale tvqrllpvlcqahgltpeqvvaias ngggsivaqlsrpdpalaaltndhl valaclggrpaldavkkglphapal ikrtnrripertshrva PKKKRKVSS dykdhdgdykdhdidykddddk AAGGGGSGRA

HHHHHH 29 dCAS9 RNA mdkkysiglaigtnsvgwavitdey dCas9 domain guided DNA kvpskkfkvlgntdrhsikknliga binding llfdsgetaeatrlkrtarrrytrr polypeptide knricylqeifsnemakvddsffhr leesflveedkkherhpifgnivde vayhekyptiyhlrkklvdstdkad lrliylalahmikfrghfliegdln pdnsdvdklfiqlvqtynqlfeenp inasgvdakailsarlsksrrlenl iaqlpgekknglfgnlialslgltp nfksnfdlaedaklqlskdtydddl dnllaqigdqyadlflaaknlsdai llsdilrvnteitkaplsasmikry dehhqdltllkalvrqqlpekykei ffdqskngyagyidggasqeefykf ikpilekmdgteellvklnredllr kqrtfdngsiphqihlgelhailrr qedfypflkdnrekiekiltfripy yvgplargnsrfawmtrkseetitp wnfeewdkgasaqsfiermtnfdkn lpnekvlpkhsllyeyftvyneltk vkyvtegmrkpaflsgeqkkaivdl lfktnrkvtvkqlkedyfkkiecfd sveisgvedrfnaslgtyhdllkii kdkdfldneenediledivltltlf edremieerlktyahlfddkvmkql krrrytgwgrlsrklingirdkqsg ktildflksdgfanmfmqlihddsl tfkediqkaqvsgqgdslhehianl agspaikkgilqtvkwdelvkvmgr hkpeniviemarenqttqkgqknsr ermkrieegikelgsqilkehpven tqlqneklylyylqngrdmyvdqel dinrlsdydvdaivpqsflkddsid nkvltrsdknrgksdnvpseevvkk mknywrqllnaklitqrkfdnltka ergglseldkagfikrqlvetrqit khvaqildsrmntkydendklirev kvitlksklvsdfrkdfqfykvrei nnyhhahdaylnavvgtalikkypk lesefvygdykvydvrkmiakseqe igkatakyffysnimnffkteitla ngeirkrpiietngetgeivwdkgr dfatvrkvlsmpqvnivkktevqtg gfskesilpkrnsdkliarkkdwdp kkyggfdsptvaysvlvvakvekgk skklksvkellgitimerssfeknp idfleakgykevkkdliiklpkysi felengrkrmlasagelqkgnelal pskyvnflylashyeklkgspedne qkqlfveqhkhyldeiieqisefsk rviladanldkvlsaynkhrdkpir eqaeniihlftltnlgapaafkyfd ttidrkrytstkevldatlihqsit glyetridlsqlggd 30 dCAS9 RNA mdkkysiglaigtnsvgwavitdey dCas9 domain is guided DNA kvpskkfkvlgntdrhsikknliga in lower case, binding llfdsgetaeatrlkrtarrrytrr SV40 NLS is polypeptide knricylqeifsnemakvddsffhr uppercase and with leesflveedkkherhpifgnivde underlined, Nuclear vayhekyptiyhlrkklvdstdkad 3xFLAG Localization lrliylalahmikfrghfliegdln sequence is lower and pdnsdvdklfiqlvqtynqlfeenp case and Transcription inasgvdakailsarlsksrrlenl underlined, the Activator iaqlpgekknglfgnlialslgltp VP64 domain is sequences nfksnfdlaedaklqlskdtydddl lower case, with C- dnllaqigdqyadlflaaknlsdai underlined, and terminal llsdilrvnteitkaplsasmikry italicized; 6xHis 6xHis dehhqdltllkalvrqqlpekykei domain at C- domain ffdqskngyagyidggasqeefykf terminus ikpilekmdgteellvklnredllr kqrtfdngsiphqihigelhailrr qedfypflkdnrekiekiltfripy yvgplargnsrfawmtrkseetitp wnfeevvdkgasaqsfiermtnfdk nlpnekvlpkhsllyeyftvynelt kvkyvtegmrkpaflsgeqkkaivd llfktnrkvtvkqlkedyfkkiecf dsveisgvedrfnaslgtyhdllki ikdkdfldneenediledivltltl fedremieerlktyahlfddkvmkq lkrrrytgwgrlsrklingirdkqs gktildflksdgfanmfmqlihdds ltfkediqkaqvsgqgdslhehian lagspaikkgilqtvkvvdelvkvm grhkpeniviemarenqttqkgqkn srermkrieegikelgsqilkehpv entqlqneklylyylqngrdmyvdq eldinrlsdydvdaivpqsflkdds idnkvltrsdknrgksdnvpseevv kkmknywrqllnaklitqrkfdnlt kaergglseldkagfikrqlvetrq itkhvaqildsrmntkydendklir evkvitlksklvsdfrkdfqfykvr ei nnyhhahdaylnavvgtalikkypkl esefvygdykvydvrkmiakseqei gkatakyffysnimnffkteitlan geirkrplietngetgeivwdkgrd fatvrkvlsmpqvnivkktevqtgg fskesilpkrnsdkliarkkdwdpk kyggfdsptvaysvlvvakvekgks kklksvkellgitimerssfeknpi dfleakgykevkkdliiklpkysif elengrkrmlasagelqkgnelalp skyvnflylashyeklkgspedneq kqlfveqhkhyldeiieqisefskr viladanldkvlsaynkhrdkpire qaeniihlftltnlgapaafkyfdt tidrkrytstkevldatlihqsitg lyetridlsalggdGS PKKKRKVSS dykdhdgdykdhdidykddddk AAGGGGSGRAd

HHHHHH 31 ODP2_promoter_c ttatttttccttcggaaatt T (Thymine) rRNA-1 encoding residues are U sequence (uracil) residues in encoded RNA 32 ODP2_promoter_c AAAATAGTTTATTTTTCCTT T (Thymine) rRNA-2 encoding residues are U sequence (uracil) residues in encoded RNA 33 ODP2_promoter_c TTGGGAACCCTGGTTTTCTA T (Thymine) rRNA-3 encoding residues are U sequence (uracil) residues in encoded RNA 34 ODP2_promoter_c AAGGGCTAGTTTGGGAACCC T (Thymine) rRNA-4 encoding residues are U sequence (uracil) residues in encoded RNA 35 ODP2_promoter_c TACATACTAAGGGCTAGTTT T (Thymine) rRNA-5 encoding residues are U sequence encoding (uracil) residues sequence in encoded RNA 36 WU S2_promoter_ AAAAGATATCGTATCACCCA T (Thymine) crRNA-1 encoding residues are U sequence (uracil) residues in encoded RNA 37 WUS2_promoter_ GCAATGCAAGAGCCAGCCCC T (Thymine) crRNA-2 encoding residues are U sequence (uracil) residues in encoded RNA 38 WUS2_promoter_ GACTCTTCCAAATTCCGAAG T (Thymine) crRNA-3 residues are U encoding (uracil) residues sequence in encoded RNA 39 WUS2_promoter_ CAGTTGCCACAAGGGGAGCC T (Thymine) crRNA-4 residues are U encoding (uracil) residues sequence in encoded RNA 40 WUS2_promoter GGCAGTTACCAGTTGCCACA T (Thymine) crRNA-5 residues are U encoding (uracil) residues sequence in encoded RNA 41 sgRNA expression aaaataaatggtaaaatgtcaaatcaaaactaggctgcagtat GmU6 promoter cassette gcagagcagagtcatgatgatactacttactacaccgattcttg in lower case; for the tgtgcagaaaaatatgttaaaataattgaatctttctctag encoded sgRNA tethering ccaaatttgacaacaatgtacaccgttcatattgagagacg target sequence is approach atgcttcttgtttgctttcggtggaagctgcatatactcaa in upper case, cattactccttcagcgagttttccaactgagtcccacatt wherein the Nx20 gcccagacctaacacggtattcttgtttataatgaaatgt sequence can be gccaccacatggattgNNNNNNNNNNNNNNNNNNNN SEQ ID NO: 31, gttttagagctagaaatagcaagttaaaataaggctagtccg 32, 33, 34, 35, 36, ttatcaacttgaaaaagtggcaccgagtcggtgctttttt 37, 38, 39, or 40; or any other crRNA; remainder of the encoded sgRNA is in lower case and underlined 42 maize opaque-2 RKRKESNRESARRSRRSRYRKKV nuclear localization signal 43 60 amino acids AGSSDDCSSAASVSLRVGSHDEPCFSGD from the GDGDWMDDVRALASFLESDEDWLRCQ maize C1 TAGQLA transcriptional activation domain 44 As Cpfl (wild MTQFEGFTNLYQVSKTLRFELIPQGKTL Acidaminococcus+L type) KHIQEQGFIEEDKARNDHYKELKPIIDRI sp. (As) dCpf1 YKTYADQCLQLVQLDWENLSAAIDSYR variants include KEKTEETRNALIEEQATYRNAIHDYFIG D917A, E1006A, RTDNLTDAINKRHAEIYKGLFKAELFNG E1028A, KVLKQLGTVTTTEFIENALLRSFDKFTT D1255A, and/or YFSGFYENRKNVFSAEDISTAIPHRIVQD N1257A amino NFPKFKENCHIFTRLITAVPSLREHFENV acid substitutions KKAIGIFVSTSIEEVFSFPFYNQLLTQTQI in the wild type DLYNQLLGGISREAGTEKIKGLNEVLNL sequence; wild- AIQKNDETAHIIASLPHRFIPLFKQILSDR type residues that NTLSFILEEFKSDEEVIQSFCKYKTLLRN can be substituted ENVLETAEALFNELNSIDLTHIFISHKKL to obtain dCpfl ETISSALCDHWDTLRNALYERRISELTG variants shown in KITKSAKEKVQRSLKHEDINLQEIISAAG bold and KELSEAFKQKTSEILSHAHAALDQPLPT underlined TLKKQEEKEILKSQLDSLLGLYHLLDWF AVDESNEVDPEFSARLTGIKLEMEPSLS FYNKARNYATKKPYSVEKFKLNFQMPT LASGWDVNKEKNNGAILFVKNGLYYL GIMPKQKGRYKALSFEPTEKTSEGFDK MYYDYFPDAAKMIPKCSTQLKAVTAHF QTHTTPILLSNNFIEPLEITKEIYDLNNPE KEPKKFQTAYAKKTGDQKGYREALCK WIDFTRDFLSKYTKTTSIDLSSLRPSSQY KDLGEYYAELNPLLYHISFQRIAEKEIM DAVETGKLYLFQIYNKDFAKGHHGKPN LHTLYWTGLFSPENLAKTSIKLNGQAEL FYRPKSRMKRMAHRLGEKMLNKKLKD QKTPIPDTLYQELYDYVNHRLSHDLSDE ARALLPNVITKEVSHEIIKDRRFTSDKFF FHVPITLNYQAANSPSKFNQRVNAYLK EHPETPIIGI D RGERNLIYITVIDSTGKILE QRSLNTIQQFDYQKKLDNREKERVAAR QAWSVVGTIKDLKQGYLSQVIHEIVDL MIHYQAVVVL E NLNFGFKSKRTGIAEK AVYQQFEKMLIDKLNCLVLKDYPAEKV GGVLNPYQLTDQFTSFAKMGTQSGFLF YVPAPYTSKIDPLTGFVDPFVWKTIKNH ESRKHFLEGFDFLHYDVKTGDFILFLFK MNRNLSFQRGLPGFMPAWDIVFEKNET QFDAKGTPFIAGKRIVPVIENHRFTGRY RDLYPANELIALLEEKGIVFRDGSNILPK LLENDDSHAIDTMVALIRSVLQMRNSN AATGEDYINSPVRDLNGVCFDSRFQNPE WPMDA D ANGAYHIALKGQLLLNHLKE SKDLKLQNGISNQDWLAYIQELRN 45 LbCpf1 (wild MSKLEKFTNCYSLSKTLRFKAIPVGKTQ Lachnospiraceae type) ENIDNKRLLVEDEKRAEDYKGVKKLLD bacterium (Lb) RYYLSFINDVLHSIKLKNLNNYISLFRK DCpf1 variants KTRTEKENKELENLEINLRKEIAKAFKG include D832A, NEGYKSLFKKDIIETILPEFLDDKDEIAL E925A, and/or VNSFNGFTTAFTGFFDNRENMFSEEAKS D1148A amino TSIAFRCINENLTRYISNMDIFEKVDAIF acid substitutions DKHEVQEIKEKILNSDYDVEDFFEGEFF in the NFVLTQEGIDVYNAIIGGFVTESGEKIKG wild type LNEYINLYNQKTKQKLPKFKPLYKQVL sequence; wild- SDRESLSFYGEGYTSDEEVLEVFRNTLN type residues that KNSEIFSSIKKLEKLFKNFDEYSSAGIFV can be substituted KNGPAISTISKDIFGEWNVIRDKWNAEY to obtain dCpf1 DDIHLKKKAVVTEKYEDDRRKSFKKIG variants SFSLEQLQEYADADLSVVEKLKEIIIQKV shown in DEIYKVYGSSEKLFDADFVLEKSLKKN bold and DAVVAIMKDLLDSVKSFENYIKAFFGE underlined GKETNRDESFYGDFVLAYDILLKVDHIY DAIRNYVTQKPYSKDKFKLYFQNPQFM GGWDKDKETDYRATILRYGSKYYLAI MDKKYAKCLQKIDKDDVNGNYEKINY KLLPGPNKMLPKVFFSKKWMAYYNPS EDIQKIYKNGTFKKGDMFNLNDCHKLI DFFKDSISRYPKWSNAYDFNFSETEKYK DIAGFYREVEEQGYKVSFESASKKEVD KLVEEGKLYMFQIYNKDFSDKSHGTPN LHTMYFKLLFDENNHGQIRLSGGAELF MRRASLKKEELWHPANSPIANKNPDN PKKTTTLSYDVYKDKRFSEDQYELHIPI AINKCPKNIFKINTEVRVLLKHDDNPYV IGI D RGERNLLYIVVVDGKGNIVEQYSL NEIINNFNGIRIKTDYHSLLDKKEKERFE ARQNWTSIENIKELKAGYISQVVHKICE LVEKYDAVIAL E DLNSGFKNSRVKVEK QVYQKFEKMLIDKLNYMVDKKSNPCA TGGALKGYQITNKFESFKSMSTQNGFIF YIPAWLTSKIDPSTGFVNLLKTKYTSIAD SKKFISSFDRIMYVPEEDLFEFALDYKN FSRTDADYIKKWKLYSYGNRIRIFRNPK KNNVFDWEEVCLTSAYKELFNKYGINY QQGDIRALLCEQSDKAFYSSFMALMSL MLQMRNSITGRTDV D FLISPVKNSDGIF YDSRNYEAQENAILPKNADANGAYNIA RKVLWAIGQFKKAEDEKLDKVKIAISN KEWLEYAQTSVKH 46 Fn Cpf1 (wild MSIYQEFVNKYSLSKTLRFELIPQGKTL Francisella type) ENIKARGLILDDEKRAKDYKKAKQIIDK novicida (Fn) YHQFFIEEILSSVCISEDLLQNYSDVYFK dCpfl variants LKKSDDDNLQKDFKSAKDTIKKQISEYI include FnCpf1 KDSEKFKNLFNQNLIDAKKGQESDLIL amino acid WLKQSKDNGIELFKANSDITDIDEALEII substitutions KSFKGWTTYFKGFHENRKNVYSSNDIP in TSIIYRIVDDNLPKFLENKAKYESLKDK the wild type APEAINYEQIKKDLAEELTFDIDYKTSE sequence; wild- VNQRVFSLDEVEEIANFNNYLNQSGITK type residues that FNTIIGGKFVNGENTKRKGINEYINLYS can be substituted QQINDKTLKKYKMSVLFKQILSDTESKS to obtain FVTDKLEDDSDVVTTMQSFYEQIAAFKT dCpf1 VEEKSIKETLSLLFDDLKAQKLDLSKIYF variants KNDKSLTDLSQQVFDDYSVIGTAVLEYI shown in TQQIAPKNLDNPSKKEQELIAKKTEKAK bold and YLSLETIKLALEEFNKHRDIDKQCRFEEI underlined LANFAAIPMIFDEIAQNKDNLAQISIKYQ NQGKKDLLQASAEDDVKAIKDLLDQT NNLLHKLKIFHISQSEDKANILDKDEHF YLVFEECYFELANIVPLYNKIRNYITQKP YSDEKFKLNFENSTLANGWDKNKEPDN TAILFIKDDKYYLGVMNKKNNKIFDDK AIKENKGEGYKKIVYKLLPGANKMLPK VFFSAKSIKFYNPSEDILRIRNHSTHTKN GSPQKGYEKFEFNIEDCRKFIDFYKQSIS KHPEWKDFGFRFSDTQRYNSIDEFYRE VENQGYKLTFENISESYIDSVVNQGKLY LFQIYNKDFSAYSKGRPNLHTLYWKAL FDERNLQDVVYKLNGEAELFYRKQSIP KKITHPAKEAIANKNKDNPKKESVFEY DLIKDKRFTEDKFFFHCPITINFKSSGAN KFNDEINLLLKEKANDVHILSI D RGERH LAYYTLVDGKGNIIKQDTFNIIGNDRMK TNYHDKLAAIEKDRDSARKDWKKINNI KEMKEGYLSQVVHEIAKLVIEYNAIVVF E DLNFGFKRGRFKVEKQVYQKL E KMLI EKLNYLWKDNEFDKTGGVLRAYQLTA PFETFKKMGKQTGIIYYVPAGFTSKICP VTGFVNQLYPKYESVSKSQEFFSKFDKI CYNLDKGYFEFSFDYKNFGDKAAKGK WTIASFGSRLINFRNSDKNHNWDTREV YPTKELEKLLKDYSIEYGHGECIKAAIC GESDKKFFAKLTSVLNTILQMRNSKTGT ELDYLISPVADVNGNFFDSRQAPKNMP QDA D A N GAYHIGLKGLMLLGRIKNNQ EGKKLNLVIKNEEYFEFVQNRNN 47 CasJ (wild type) MQQYQVSKTVRFGLTLKNSEKKHATH dCasJ variants LLLKDLVNVSEERIKNEITKDDKNQSEL include D901A SFFNEVIETLDLMDKYIKDWENCFYRT and/or El228A DQIQLTKEYYKVIAKKACFDWFWTND amino acid RGMKFPTSSIISFNSLKSSDKSKTSDNLD substitutions in RKKKILDYWKGNIFKTQKAIKDVLDITE the wild type DIQKAIEEKKSHREINRVNHRKMGIHLI sequence; wild- HLINDTLVPLCNGSIFFGNISKLDFCESE type residues NEKLIDFASTEKQDERKFLLSKINEIKQY that can be FEDNGGNVPFARATLNRHTANQKPDRY substituted NEEIKKLVNELGVNSLVRSLKSKTIEEIK to obtain THFEFENKNKINELKNSFVLSIVEKIQLF dCasJ KYKTIPASVRFLLADYFEEQKLSTKEEA variants LTIFEEIGKPQNIGFDYIQLKEKDNFTLK shown in KYPLKQAFDYAWENLARLDQNPKANQ bold and FSVDECKRFFKEVFSMEMDNINFKTYA underlined LLLALKEKTTAFDKKGEGAAKNKSEIIE QIKGWEELDQPFKIIANTLREEVIKKED ELNVLKRQYRETDRKIKTLQNEIKKIKN QIKNLENSKKYSFPEIIKWIDLTEQEQLL DKNKQAKSNYQKAKGDLGLIRGSQKTS INDYFYLTDKVYRKLAQDFGKKMADL REKLLDKNDVNKIKYLSYIVKDNQGYQ YTLLKPLEDKNAEIIELKSEPNGDLKLFE IKSLTSKTLNKFIKNKGAYKEFHSAEFE HKKIKEDWKNYKYNSDFIVKLKKCLSH SDMANTQNWKAFGWDLDKCKSYETIE KEIDQKSYQLVEIKLSKTTIEKWVKENN YLLLPIVNQDITAEKLKVNTNQFTKDW QHIFEKNPNHRLHPEFNIAYRQPTKDYA KEGEKRYSRFQLTGQFMYEYIPQDANY ISRKEQITLFNDKEEQKIQVETFNNQIAK ILNAEDFYVIGI D  RGITQLATLCVLNKN GVIQGGFEIFTREFDYTNKQWKHTKLK ENRNILDISNLKVETTVNGEKVLVDLSE VKTYLRDENGEPMKNEKGVILTKDNLQ KIKLKQLAYDRKLQYKMQHEPELVLSF LDRLENKEQIPNLLASTKLISAYKEGTA YADIDIEQFWNILQTFQTIVDKFGGIENA KKTMEFRQYTELDASFDLKNGVVANM VGVVKFIMEKYNYKTFIAL E DLTFAFG QSIDGINGERLRSTKEDKEVDFKEQENS TLAGLGTYHFFEMQLLKKLSKTQIGNEI KHFVPAFRSTENYEKIVRKDKNVKAKI VSYPFGIVSFVNPRNTSISCPNCKNANK SNRIKKENDRILCKHNIEKTKGNCGFDT ANFDENKLRAENKGKNFKYISSGDANA AYNIAVKLLEDKIFEINKK 48 Zinc finger linker TGEKP peptide 49 SV40 large T PKKKRKV antigen NLS 50 Class II K(K/R)X(K/R) monopartite NLS consensus 51 Bipartite NLS (K/R)(K/R)X₁₀₋₁₂(K/R)_(3/5) where K/R)3/5 consensus represents at least three of either lysine or arginine of five consecutive amino acids 52 Class 5 Plant NLS LGKR(K/R)(W/F/Y) 53 (Gly4Ser)n GGGGS n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 54 Ser(Gly4Ser)n SGGGGS n is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 55 cell-penetrating YGRKKRRQRRR peptide (CPP) 56 cell-penetrating RRQRRTSKLMKR peptide (CPP) 57 cell-penetrating GWTLNSAGYLLGKINLKALAALAKKIL peptide (CPP) 58 cell-penetrating KALAWEAKLAKALAKALAKHLAKALA peptide (CPP) KALKCEA 59 cell-penetrating RQIKIWFQNRRMKWKK peptide (CPP) 60 cell-penetrating YGRKKRRQRRR peptide (CPP) 61 cell-penetrating RKKRRQRR peptide (CPP) 62 cell-penetrating YARAAARQARA peptide (CPP) 63 cell-penetrating THRLPRRRRRR peptide (CPP) 64 cell-penetrating GGRRARRRRRR peptide (CPP) 65 ZmWUS2-TALE- TGGCCCTCTGCATCCTCCTCATGATAG 1 binding site CT 66 ZmWUS2-TALE- TCTTCCAAATTCCGAAGCGGCCAATG 2 binding site CAAT 67 ZmWUS2-TALE- TATCGTATCACCCATGGCCATGACCCC 3 binding site CCT 68 ZmODP2-TALE-1 TATTCCTAATATATATATCATACTCTC binding site CAT 69 ZmODP2-TALE-2 TCTTCTGGAGTGTACCAGTTGTATAAA binding site TAT 70 ZmODP2-TALE-3 TGCACTGTCCAAAATGGCTTCCTGATC binding site CCCT 71 Conserved Maize GTACTCATATATGGGCACACATATAG ODP2 promoter ACATGTTTTGAGGAAAATGAGACAAA GTATAGTGGAGACTTCCCTAGAAAGC AGAAGAAAAAGAAGTGGTTTATGTTC CGTTAAATCATACTACAACTTTTTTTT ATTATACTCTCCATTTTGTCATCATTA GGTACTCATATATGGGCACACATATA GTACTGCCAATTTTTCTTGCTAAAAAA AGTTCCACTATATATATGTATGTATGC ACAAATAAACTAATTTTCTTAGAAAA GAAAACCGGTGTAATACATACTAAGG GCTAGTTTGGGAACCCTGGTT 72 ZmWUS2-TALE- msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of 1b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVD akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lower RVD, and C- stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmi case; truncated terminal aalpeatheaivgvgkqwsgaralealltvageirgpplqldt TAL terminal domain gqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upper iasnhggkqaletvqrllpvlcqahgltpeqvvaiasnhggk case and qaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrl underlined Ipvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqah gltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvv aiasngggkqaletvqrllpvlcqahgltpeqvvaiashdgg kqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvq rllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcqa hgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqv vaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngg gkqaletvqrllpvlcqahgltpeqvvaiashdggkqaletv qrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcq ahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeq vvaiashdggkqaletvqrllpvlcqahgltpeqvvaiashd ggkqaletvqrllpvlcqahgasqltpeqvvaiasnggg RPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNR RIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRR VGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSL ERDLDAPSPMHEGDQTRAS 73 ZmWUS2-TALE- MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- lb ATF Ifntslfdslppfgahhteaatgewdevqsglraadappptm terminus residues rvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case; qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus of vavkyqdmiaalpeatheaivgvgkqwsgaralealltva TALE, RVD gelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C- nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lower qvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiash case; truncated dggkqaletvqrllpvlcqahgltpeqvvaiashdggkqale TAL terminal tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl domain in upper cqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltp case and eqvvaiashdggkqaletvqrllpvlcqahgltpeqvvaias underlined, ngggkqaletvqrllpvlcqahgltpeqvvaiasnhggkqa extended SV40 letvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllp Nuclear vlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahglt Localization peqvvaiasngggkqaletvqrllpvlcqahgltpeqvvaia Sequence in shdggkqaletvqrllpvlcqahgltpeqvvaiashdggkq lowercase, double aletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrll underlined, pvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahg italics; VP64 ltpeqvvaiashdggkqaletvqrllpvlcqahgasqltpeq transcriptional vvaiasnggg activation domain RPALESIVAQLSRPDPALAAL is lowercase, TNDHLVALACLGGRPALDAVKKGLPH underlined APALIKRTNRRIPERTSHRVADHAQVVR VLGFFQCHSHPAQAFDDAMTQFGMSR HGLLQLFRRVGVTELEARSGTLPPASQR WDRILQASGMKRAKPSPTSTQTPDQAS LHAFADSLERDLDAPSPMHEGDQTRAS

GRAdalddfdldml ssdalddfdldmlgsdalddfdldmlg sdalddfdldml 74 ZmWUS2-TALE- TGGCCCTCTGCATCCTCCT lb binding site 75 ZmWUS2-TALE- msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of 2b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVD RVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lower terminal domain stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmi case; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminal gqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upper iasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggk case and qaletvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrl underlined lpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqah gltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvv aiasniggkqaletvqrllpvlcqahltpeqvvaiasniggkq aletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrll pvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahg ltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvva iashdggkqaletvqrllpvlcqahgltpeqvvaiasniggk qaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrl lpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqah gltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvv aiasnhggkqaletvqrllpvlcqahgasqltpeqvvaiasn gggRPALESIVAQLSRPDPALAALTNDHL VALACLGGRPALDAVKKGLPHAPALIK RTNRRIPERTSHRVADHAQVVRVLGFF QCHSHPAQAFDDAMTQFGMSRHGLLQ LFRRVGVTELEARSGTLPPASQRWDRIL QASGMKRAKPSPTSTQTPDQASLHAFA DSLERDLDAPSPMHEGDQTRAS 76 ZmWUS2-TALE- MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 2b ATF lfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residues rvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case; qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus of vavkyqdmiaaipeatheaivgvgkqwsgaralealltva TALE, RVD gelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C- nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lower qvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasn case; truncated hggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale TAL terminal tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl domain in upper cqahgltpeqvvaiasniggkqaletvqrllpvlcqahltpeq case and vvaiasniggkqaletvqrllpvlcqahgltpeqvvaiashd underlined, ggkqaletvqrllpvlcqahgltpeqvvaiasngggkqalet extended SV40 vqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlc Nuclear qahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpe Localization qvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiash Sequence in dggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale lowercase, double tvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvl underlined, cqahgltpeqvvaiasnhggkqaletvqrllpvlcqahgas italics; VP64 ciltDecivvaiasngggRPALESIVAQLSRPDPA transcriptional LAALTNDHLVALACLGGRPALDAVKK activation domain GLPHAPALIKRTNRRIPERTSHRVADHA is lowercase, QVVRVLGFFQCHSHPAQAFDDAMTQF underlined GMSRHGLLQLFRRVGVTELEARSGTLP PASQRWDRILQASGMKRAKPSPTSTQT PDQASLHAFADSLERDLDAPSPMHEGD QTRAS

GRAdalddfdldmlgsd alddfdldmlRsdalddfdldmlgsdalddfdldml 77 ZmWUS2-TALE- TGTGTCAACTTCACTTGT 2b binding site 78 ZmWUS2-TALE- msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of 3b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVD RVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lower terminal domain stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmi case; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminal gqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upper iashdggkqaletvqrllpvlcqahgltpeqvvaiasnhggk case and qaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrl underlined lpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqah gltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqvv aiashdggkqaletvqrllpvlcqahgltpeqvvaiasnigg kqaletvqrllpvlcqahgltpeqvvaiashdggkqaletvq rllpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqa hgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqv vaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngg gkqaletvqrllpvlcqahgltpeqvvaiasnhggkqaletv qrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlcq ahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeq vvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasni ggkqaletvqrllpvlcqahgasqltpeqvvaiasnggg RPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNR RIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRR VGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSL ERDLDAPSPMHEGDQTRAS 79 ZmWUS2-TALE- MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 3b ATF lfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residues rvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case; qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus of vavkyqdmiaalpeatheaivgvgkqwsgaraleal1tva TALE, RVD gelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C- nltetltpeqvvaiashdggkqaletvqrllpvlcqahgltpe terminus in lower qvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasn case; truncated gggkqaletvqrllpvlcqahgltpeqvvaiasniggkqale TAL terminal tvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvl domain in upper cqahgltpeqvvaiashdggkqaletvqrllpvlcqahgltp case and eqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaias underlined, hdggkqaletvqrllpvlcqahgltpeqvvaiashdggkqa extended SV40 letvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllp Nuclear vlcqahgltpeqvvaiasniggkqaletvqrllpvlcqahglt Localization peqvvaiasngggkqaletvqrl1pvlcqahgltpeqvvaia Sequence in snhggkqaletvqrllpvlcqahgltpeqvvaiasnhggkq lowercase, double aletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrll underlined, pvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahg italics; VP64 ltpeqvvaiasniggkqaletvqrllpvlcqahgasqltpeqv transcriptional vaiasnggg activation domain RPALESIVAQLSRPDPALAAL is lowercase, TNDHLVALACLGGRPALDAVKKGLPH underlined APALIKRTNRRIPERTSHRVADHAQVVR VLGFFQCHSHPAQAFDDAMTQFGMSR HGLLQLFRRVGVTELEARSGTLPPASQR WDRILQASGMKRAKPSPTSTQTPDQAS LHAFADSLERDLDAPSPMHEGDQTRAS

GRAdalddfdldml ssdalddfdldmlgsdalddfdldmlg sdalddfdldml 80 ZmWUS2-TALE- TCGTATCACCCATGGCCAT 3b binding site 81 ZmODP2-TALE- msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of 1b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVD RVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lower terminal domain stvaqhhealvghgfithahivalsqhpaalgtvavkyqdmi case; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminal gqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upper iashdggkqaletvqrllpvlcqahgltpeqvvaiashdggk case and qaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrl underlined lpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqah gltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqw aiasngggkqaletvqrllpvlcqahgltpeqvvaiasnigg kqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvq rllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqa hgltpeqvvaiasngggkqaletvqrllpvlcqahgltpeqv vaiasniggkqaletvqrllpvlcqahgltpeqvvaiasngg gkqaletvqrllpvlcqahgltpeqvvaiasniggkqaletv qrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcq ahgltpeqvvaiashdggkqaletvqrllpvlcqahgltpeq vvaiasniggkqaletvqrllpvlcqahgasqltpeqvvaias nggg RPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNR RIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRR VGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSL ERDLDAPSPMHEGDQTRAS 82 ZmODP2-TALE- MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 1b ATF lfntslfdslppfgahhteaatgewdevqsglraadappptm terminus rvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq residues qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt 2-7 in upper case; vavkyqdmiaalpeatheaivgvgkqwsgaralealltva N terminus of gelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl TALE, RVD nltetltpeqvvaiashdggkqaletvqrllpvlcqahgltpe domain, C- qvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasn terminus gggkqaletvqrllpvlcqahgltpeqvvaiasniggkqale in lower tvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlc case; qahgltpeqvvaiasngggkqaletvqrllpvlcqahgltpe truncated qvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasn TAL terminal gggkqaletvqrllpvlcqahgltpeqvvaiasniggkqale domain tvqrllpvlcqahgltpeqvvaiasngggkqaletvqrllpvl in upper cqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpe case and qvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasn underlined, iggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale extended SV40 tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl Nuclear cqahgltpeqvvaiasniggkqaletvqrllpvlcqahgasq Localization ltpeciwaiasnggg Sequence in RPALESIVAQLSRPDPALAAL lowercase, double TNDHLVALACLGGRPALDAVKKGLPH underlined, APALIKRTNRRIPERTSHRVADHAQVVR italics; VP64 VLGFFQCHSHPAQAFDDAMTQFGMSR transcriptional HGLLQLFRRVGVTELEARSGTLPPASQR activation domain WDRILQASGMKRAKPSPTSTQTPDQAS is lowercase, LHAFADSLERDLDAPSPMHEGDQTRAS underlined

GRA dalddfdldmlgsdalddfdldmlg sdalddfdldmlgsdalddfdldml 83 ZmODP2-TALE- TCCTAATATATATATCAT 1b binding site 84 ZmODP2-TALE- msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of 2b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVD RVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lower terminal stvaqhhealvghgfthahivalsqhpaalgtvavkyqdmi case; truncated domain aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminal gqllkiakrggvtaveavhawrnaltgaplnltetltpeqvva domain in upper iasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggk case and qaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrll underlined pvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqahg ltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvva iasniggkqaletvqrllpvlcqahgltpeqvvaiasnhggk qaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrl lpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqah gltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvv aiasngggkqaletvqrllpvlcqahgltpeqvvaiasnigg kqaletvqrllpvlcqahgltpeqvvaiasngggkqaletvq rllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlcqa hgltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqv vaiasniggkqaletvqrllpvlcqahgasqltpeqvvaiasn gggRPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNR RIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRR VGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSL ERDLDAPSPMHEGDQTRAS 85 ZmODP2-TALE- MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 2b ATF lfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residues rvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case; qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus of vavkyqdmiaalpeatheaivgvgkqwsgaralealltva TALE, RVD gelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C- nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lower qvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiasn case; truncated iggkqaletvqrllpvlcqahgltpeqvvaiashdggkqale TAL terminal tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl domain in upper cqahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpe case and qvvaiasnhggkqaletvqrllpvlcqahgltpeqvvaiasn underlined, gggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale extended SV40 tvqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvl Nuclear cqahgltpeqvvaiasngggkqaletvqrllpvlcqahgltp Localization eqvvaiasniggkqaletvqrllpvlcqahgltpeqvvaias Sequence in ngggkqaletvqrllpvlcqahgltpeqvvaiasniggkqal lowercase, double etvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvl underlined, cqahgltpeqvvaiasniggkqaletvqrllpvlcqahgasq italics; VP64 ltpeqvvaiasnggg transcriptional RPALESIVAQLSRPDPALAAL activation domain TNDHLVALACLGGRPALDAVKKGLPH is lowercase, APALIKRTNRRIPERTSHRVADHAQVVR underlined VLGFFQCHSHPAQAFDDAMTQFGMSR HGLLQLFRRVGVTELEARSGTLPPASQR WDRILQASGMKRAKPSPTSTQTPDQAS LHAFADSLERDLDAPSPMHEGDQTRAS

GRAdalddfdldml ssdalddfdldmlgsdalddfdldmlg sdalddfdldml 86 ZmODP2-TALE- TGTACCAGTTGTATAAAT 2b binding site 87 ZmODP2-T ALE- msrtrlpsppapspafsadsfsdllrqfdpslfntslfdslppf N terminus of 3b N-terminus, gahhteaatgewdevqsglraadappptmrvavtaarppr TALE and RVD RVD, and C- akpaprrraaqpsdaspaaqvdlrtlgysqqqqekikpkvr domain in lower terminal domain stvaqhhealvghgfithahivalsqhpaalgtvavkyqdmi case; truncated aalpeatheaivgvgkqwsgaralealltvagelrgpplqldt TAL terminal gqllkiakrggvtaveavhawmaltgaplnltetltpeqvva domain in upper iasnhggkqaletvqrllpvlcqahgltpeqvvaiasngggk case and qaletvqrllpvlcqahgltpeqvvaiashdggkqaletvqrl underlined lpvlcqahgltpeqvvaiashdggkqaletvqrllpvlcqah gltpeqvvaiasniggkqaletvqrllpvlcqahgltpeqvv aiasniggkqaletvqrllpvlcqahgltpeqvvaiasniggk qaletvqrllpvlcqahgltpeqvvaiasniggkqaletvqrll pvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqahg ltpeqvvaiasnhggkqaletvqrllpvlcqahgltpeqvva iasnhggkqaletvqrllpvlcqahgltpeqvvaiashdggk qaletvqrllpvlcqahgltpeqvvaiasngggkqaletvqrl lpvlcqahgltpeqvvaiasngggkqaletvqrllpvlcqah gltpeqvvaiashdggkqaletvqrllpvlcqahgltpeqvv aiashdggkqaletvqrllpvlcqahgasqltpeqvvaiasn gggRPALESIVAQLSRPDPALAALTNDHLVALA CLGGRPALDAVKKGLPHAPALIKRTNR RIPERTSHRVADHAQVVRVLGFFQCHS HPAQAFDDAMTQFGMSRHGLLQLFRR VGVTELEARSGTLPPASQRWDRILQAS GMKRAKPSPTSTQTPDQASLHAFADSL ERDLDAPSPMHEGDQTRAS 88 ZmODP2-TALE- MHHHHHHmsrtrlpsppapspafsadsfsdllrqfdps 6xHis tag at N- 3b ATF lfntslfdslppfgahhteaatgewdevqsglraadappptm terminus residues rvavtaarpprakpaprrraaqpsdaspaaqvdlrtlgysqq 2-7 in upper case; qqekikpkvrstvaqhhealvghgfthahivalsqhpaalgt N terminus of vavkyqdmiaalpeatheaivgvgkqwsgaralealltva TALE, RVD gelrgpplqldtgqllkiakrggvtaveavhawrnaltgapl domain, C- nltetltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe terminus in lower qvvaiasngggkqaletvqrllpvlcqahgltpeqvvaiash case; truncated dggkqaletvqrllpvlcqahgltpeqvvaiashdggkqale TAL terminal tvqrllpvlcqahgltpeqvvaiasniggkqaletvqrllpvlc domain in upper qahgltpeqvvaiasniggkqaletvqrllpvlcqahgltpe case and qvvaiasniggkqaletvqrllpvlcqahgltpeqvvaiasni underlined, SV40 ggkqaletvqrllpvlcqahgltpeqvvaiasngggkqalet Nuclear vqrllpvlcqahgltpeqvvaiasnhggkqaletvqrllpvlc Localization qahgltpeqvvaiasnhggkqaletvqrllpvlcqahgltpe Sequence in qvvaiashdggkqaletvqrllpvlcqahgltpeqvvaiasn lowercase, double gggkqaletvqrllpvlcqahgltpeqvvaiasngggkqale underlined, tvqrllpvlcqahgltpeqvvaiashdggkqaletvqrllpvl italics; VP64 cqahgltpeqvvaiashdggkqaletvqrllpvlcqahgas transcriptional qltpeqvvaiasnggg activation domain RPALESIVAQLSRPDPA is lowercase, LAALTNDHLVALACLGGRPALDAVKK underlined GLPHAPALIKRTNRRIPERTSHRVADHA QVVRVLGFFQCHSHPAQAFDDAMTQF GMSRHGLLQLFRRVGVTELEARSGTLP PASQRWDRILQASGMKRAKPSPTSTQT PDQASLHAFADSLERDLDAPSPMHEGD QTRAS

GRAdalddfdldmlgsdalddfdldml gsdalddfdldmlgsdalddfdldml 89 ZmODP2-TALE- TGTCCAAAATGGCTTCCT 3b binding site 90 dCAS9 RNA MHHHHHHmdkkysiglaigtnsvgwa 6xHis tag at N- guided DNA vitdeykvpskkfkvlgntdrhsik terminus residues binding knligallfdsgetaeatrlkrtar 2-7 in upper case; polypeptide with rrytrrknricylqeifsnemakvd dCas9 domain in Nuclear dsffhrleesflveedkkherhpif lowercase; Localization and gnivdevayhekyptiyhlrkklvd extended SV40 Transcription stdkadlrliylalahmikfrghfl Nuclear Activator iegdlnpdnsdvdklfiqlvqtynq Localization sequences with N- lfeenpinasgvdakailsarisks Signal in terminal 6xHis rrlenliaqlpgekknglfgnlial lowercase, domain sigltpnfksnfdlaedaklqlskd underlined; VP64 tydddldnllaqigdqyadlflaak transcriptional nlsdaillsdilrvnteitkaplsa activation domain smikrydehhqdltllkalvrqqlp in lowercase, ekykeiffdqskngyagyidggasq italics, and double eefykfikpilekmdgteellvkln underlined redllrkqrtfdngsiphqihlgel hailrrqedfypflkdnrekiekil tfripyyvgplargnsrfawmtrks eetitpwnfeevvdkgasaqsfier mtnfdknlpnekvlpkhsllyeyft vyneltkvkyvtegmrkpaflsgeq kkaivdllfktnrkvtvkqlkedyf kkiecfdsveisgvedrfnaslgty hdllkiikdkdfldneenediledi vltltlfedremieerlktyahlfd dkvmkqlkmytgwgrlsrklingir dkqsgktildflksdgfanmfmqli hddsltfkediqkaqvsgqgdslhe hianlagspaikkgilqtvkvvdel vkvmgrhkpeniviemarenqttqk gqknsrermkrieegikelgsqilk ehpventqlqneklylyylqngrdm yvdqeldinrlsdydvdaivpqsfl kddsidnkvltrsdknrgksdnvps eevvkkmknywrqllnaklitqrkf dnltkaergglseldkagfikrqlv etrqitkhvaqildsrmntkydend klirevkvitlksklvsdfrkdfqf ykvreinnyhhahdaylnavvgtal ikkypklesefvygdykvydvrkmi akseqeigkatakyffysnimnffk teitlangeirkrplietngetgei vwdkgrdfatvrkvlsmpqvnivkk tevqtggfskesilpkrnsdkliar kkdwdpkkyggfdsptvaysvlvva kvekgkskklksvkellgitimers sfeknpidfleakgykevkkdliik lpkyslfelengrkrmlasagelqk gnelalpskyvnflylashyeklkg spedneqkqlfveqhkhyldeiieq isefskrviladanldkvlsaynkh rdkpireqaeniihlftltnlgapa afkyfdttidrkrytstkevldat lihqsitglvetridlsqlggdGSp kkkrkvSSAAGGGGSGRA

91 VPR (VP64-p65- EASGSGRADALDDFDLDMLGSDALDDF Rta) tripartite DLDMLGSDALDDFDLDMLGSDALDDF transcriptional DLDMLINSRSSGSPKKKRKVGSQYLPD activator TDDRHRIEEKRKRTYETFKSIMKKSPFS GPTDPRPPPRRIAVPSRSSASVPKPAPQP YPFTSSLSTINYDEFPTMVFPSGQISQAS ALAPAPPQVLPQAPAPAPAPAMVSALA QAPAPVPVLAPGPPQAVAPPAPKPTQA GEGTLSEALLQLQFDDEDLGALLGNST DPAVFTDLASVDNSEFQQLLNQGIPVAP HTTEPMLMEYPEAITRLVTGAQRPPDPA PAPLGAPGLPNGLLSGDEDFSSIADMDF SALLGSGSGSRDSREGMFLPKPEAGSAI SDVFEGREVCQPKRIRPFHPPGSPWANR PLPASLAPTPTGP VHEPVGSLTPAPVPQPLDPAPAVTPEAS HLLEDPDEETSQAVKALREMADTVIPQ KEEAAICGQMDLSHPPPRGHLDELTTTL ESMTEDLNLDSPLTPELNEILDTFLNDE CLLHAMHISTGLSIFDTSLF 92 Extended SV40 ASPKKKRKVEASGS Nuclear Localization Domain 93 ZnFng-WUS1 MgPKKKRKVgrlepgekpykcpecgksf SV40 Nuclear ATF srsdklvrhqrthtgekpykcpecgksfs Localization qssnlvrhqrthtgekpykcpecgksfsr Sequence in sddlvrhqrthtgekpykcpecgksfstsg uppercase, slvrhqrthtgekpykcpecgksfsredn underlined; VP64 lhthqrthtgekpykcpecgksfsdpgn transcriptional lvrharthtgaaa activation domain

is lowercase,

double underlined, italics 94 ZnFng-WUS1 ATGGGCCCTAAGAAGAAGCGCAAGGT ATF Coding CGGACGCCTTGAGCCCGGCGAAAAGC CATATAAATGCCCAGAGTGTGGCAAG AGCTTCAGCCGCAGCGACAAGCTTGT TCGCCATCAACGCACTCACACCGGCG AGAAGCCTTACAAGTGTCCAGAGTGC GGCAAGAGCTTCAGCCAGAGCAGCAA CCTCGTTCGCCATCAAAGGACCCACA CTGGAGAGAAACCATATAAGTGCCCA GAATGCGGAAAAAGCTTCTCCCGCAG CGATGATCTCGTCCGCCACCAGAGGA CTCACACCGGAGAGAAGCCTTATAAG TGCCCAGAGTGTGGCAAGTCCTTCTCC ACCAGCGGAAGCCTCGTTCGCCATCA GCGCACCCATACTGGAGAAAAACCTT ACAAGTGCCCAGAGTGTGGAAAAAGC TTTAGCCGCGAGGACAATCTCCATAC CCATCAACGCACCCACACTGGCGAAA AGCCTTACAAATGTCCAGAGTGCGGC AAGTCCTTTTCCGACCCCGGCAACCTT GTTAGGCATCAAAGGACTCATACCGG AGCTGCCGCTGACGCTCTTGACGATTT TGATCTCGACATGCTCGACGCTCTCGA TGATTTCGACCTTGACATGCTTGATGC CCTCGACGATTTCGATCTCGATATGCT TGATGCTCTTGACGACTTCGACCTTGA TATGCTCTGA 95 ZnFng-WUS2 mgPKKKRKVgrlepgekpvkcpecgks SV40 Nuclear ATF fsdpgalvrhqrthtgekpykcpecgk Localization sfsrsdnlvrhqrthtgekpykcpecg Sequence in ksfsqsgdlrrhqrthtgekpykcpecg uppercase, ksfstsgnlvrhqrthtgekpykcpecg underlined; VP64 ksfsrsdnlvrhqrthtgekpykcpecg transcriptional ksfsrsdnlvrhqrthtgaaa activation domain

is lowercase,

double underlined, italics 96 ZnFng-WUS2 ATGGGACCAAAAAAGAAGCGCAAGG ATF coding TCGGACGCCTTGAGCCCGGCGAAAAG CCATATAAGTGCCCAGAGTGCGGCAA GTCCTTCTCCGATCCCGGCGCCCTCGT TAGGCATCAGAGGACCCACACTGGAG AGAAGCCATACAAGTGTCCAGAGTGC GGAAAAAGCTTCAGCCGCTCCGACAA CCTCGTCCGCCATCAGCGCACCCACA CCGGAGAGAAACCATACAAGTGCCCA GAGTGCGGAAAGTCCTTCAGCCAGTC CGGAGATCTTCGCCGCCATCAAAGGA CTCATACCGGCGAGAAGCCTTATAAA TGCCCAGAGTGTGGAAAATCCTTCAG CACCAGCGGCAATCTCGTTCGCCACC AGAGGACTCACACTGGCGAGAAGCCA TATAAATGTCCAGAATGTGGCAAAAG CTTTTCTCGCTCCGATAACCTCGTTCG CCACCAACGCACCCATACTGGAGAAA AACCTTACAAATGCCCAGAGTGTGGA AAGAGCTTCTCTCGCAGCGACAACCT TGTCCGCCACCAGCGCACTCATACTG GAGCTGCCGCCGACGCCCTCGACGAT TTCGACCTCGATATGCTTGACGCCCTC GATGATTTCGACCTTGATATGCTTGAT GCCCTCGATGACTTCGATCTCGACATG CTCGACGCCCTTGACGACTTTGATCTC GATATGCTCTGA 97 ZnFng-WUS3 mgPKKKRKVgrlepgekpykcpecgksf SV40 Nuclear ATF sqlahlrahqrthtgekpykcpecgksf Localization sqsgdlrrhqrthtgekpykcpecgksf Sequence in sttgnltvhqrthtgekpykcpecgksf uppercase, sdcrdlarhqrthtgekpykcpecgksf underlined; VP64 srsddlvrhqrthtgekpykcpecgksf transcriptional sqssnlvrhqrthtgaaa activation domain

is lowercase,

double underlined, italics 98 ZnFng-WUS3 ATGGGCCCTAAAAAGAAGCGCAAGGT ATF coding CGGACGCCTTGAGCCCGGCGAAAAAC CTTACAAGTGCCCAGAATGCGGAAAA TCCTTTAGCCAGCTCGCCCACCTTCGC GCCCATCAGCGCACTCATACCGGAGA GAAGCCATATAAATGCCCAGAGTGTG GAAAGTCCTTCTCCCAGAGCGGCGAT CTTCGCCGCCACCAGCGCACCCACAC TGGAGAAAAACCTTATAAGTGTCCAG AATGCGGCAAGAGCTTCAGCACCACC GGCAACCTCACCGTTCACCAGAGGAC CCATACCGGCGAGAAGCCATACAAGT GTCCAGAGTGTGGCAAAAGCTTCAGC GACTGCCGCGATCTTGCTCGCCATCA AAGGACTCATACTGGAGAGAAGCCTT ACAAGTGTCCAGAGTGCGGCAAGTCC TTCAGCCGCTCCGATGACCTCGTTCGC CATCAGCGCACCCACACCGGCGAAAA GCCATATAAGTGTCCAGAGTGCGGAA AGAGCTTTTCCCAGAGCAGCAACCTT GTTAGGCACCAACGCACCCATACTGG AGCTGCCGCTGATGCTCTCGACGACTT CGACCTCGACATGCTTGACGCTCTCG ACGATTTCGATCTTGATATGCTTGATG CCCTCGATGATTTCGATCTCGACATGC TCGATGCTCTCGATGATTTTGACCTTG ACATGCTTTGA 99 ZnFng-WUS4 mgPKKKRKVgrlepgekpykcpecgksf SV40 Nuclear ATF srsdklvrhqrthtgekpykcpecgksf Localization stsgslvrhqrthtgekpykcpecgksf Sequence in srednlhthqrthtgekpykcpecgksf uppercase, sqkssliahqrthtgekpykcpecgksf underlined; VP64 srsdelvrhqrthtgekpykcpecgksf transcriptional srednlvrhqrthtgaaa activation domain

is lowercase,

double underlined, italics 100 ZnFng-WUS4 ATGGGACCAAAGAAGAAAAGGAAGG ATF coding TCGGCCGCCTTGAGCCCGGCGAAAAG CCTTATAAGTGTCCAGAGTGTGGAAA ATCCTTCTCTCGCAGCGATAAGCTCGT TAGGCACCAACGCACCCATACTGGCG AAAAACCATATAAGTGCCCAGAGTGT GGAAAGTCCTTTAGCACTAGCGGCAG CCTTGTTAGGCACCAGCGCACCCACA CCGGCGAAAAGCCTTACAAGTGTCCA GAATGTGGCAAGAGCTTCTCCCGCGA GGATAATCTCCACACTCATCAGCGCA CCCATACCGGCGAGAAACCTTACAAG TGTCCAGAATGCGGAAAAAGCTTCAG CCAAAAAAGCAGCCTCATCGCTCATC AGAGGACTCATACCGGAGAGAAGCCT TATAAATGCCCAGAGTGCGGAAAATC CTTCAGCCGCAGCGACGAACTCGTCC GCCATCAACGCACTCACACCGGAGAA AAACCATACAAATGTCCAGAGTGCGG CAAGTCCTTTAGCCGCGAGGACAACC TCCACACCCATCAAAGGACCCACACT GGAGCCGCTGCTGATGCCCTCGACGA CTTCGATCTTGACATGCTTGATGCTCT CGATGATTTCGATCTCGACATGCTTGA CGCCCTCGACGATTTCGACCTCGATAT GCTCGACGCCCTTGACGACTTTGACCT TGATATGCTCTGA 101 ZnFng-WUS1- GACTAGGTTGCGGAAGGG binding site in maize WUS2 promoter 102 ZnFng-WUS2- GAGGAGGATGCAGAGGTC binding site (minus strand) in maize WUS2 promoter 103 ZnFng-WUS3- GAAGCGGCCAATGCAAGA binding site in maize WUS2 promoter 104 ZnFng-WUS4- TAGGTGATATAGGTTGGG binding site (minus strand) in maize WUS2 promoter 105 ZnFng-WUS1 mggrlepgekpykcpecgksfsrsdklvrhqrthtgekpy Binds SEQ ID AZF DNA kcpecgksfsqssnlvrhqrthtgekpykcpecgksfsrsd NO: 101 BINDING dlvrhqrthtgekpykcpecgksfstsgslvrhqrthtgekp DOMAIN ykcpecgksfsrednlhthqrthtgekpykcpecgksfsdp gnlvrhqrthtgaaa 106 ZnFng-WUS2 mggrlepgekpykcpecgksfsdpgalvrhqrthtgekpy Binds SEQ ID AZF DNA kcpecgksfsrsdnlvrhqrthtgekpykcpecgksfsqsg NO: 102 BINDING dlrrhqrthtgekpykcpecgksfstsgnlvrhqrthtgekp DOMAIN ykcpecgksfsrsdnlvrhqrthtgekpykcpecgksfsrs dnlvrhqrthtgaaa 107 ZnFng-WUS3 mggrlepgekpykcpecgksfsqlahlrahqrthtgekpy Binds SEQ ID AZNDNA kcpecgksfsqsgdlrrhqrthtgekpykcpecgksfsttgn NO: 103 BINDING ltvhqilhtgekpykcpecgksfsdcrdlarhqrthtgekpy DOMAIN kcpecgksfsrsddlvrhqrthtgekpykcpecgksfsqss nlvrhqrthtgaaa 108 ZnFng-WUS4 mggrlepgekpykcpecgksfsrsdklvrhqrthtgekpy Binds SEQ ID AZF DNA kcpecgksfstsgslvrhqrthtgekpykcpecgksfsredn NO: 104 BINDING Ihthqrthtgekpykcpecgksfsqkssliahqrthtgekpy DOMAIN kcpecgksfsrsdelvrhqrthtgekpykcpecgksfsred nlhthqrthtgaaa

The breadth and scope of the present disclosure should not be limited by any of the above-described Examples, but should be defined only in accordance with the preceding embodiments, the following claims, and their equivalents.

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What is claimed is:
 1. A maize plant cell comprising at least one exogenous gene transcription agent that comprises the polypeptide of SEQ ID NO:93 and stimulates transcription of the endogenous WUS2 gene, wherein expression of the endogenous WUS2 polypeptide is increased in comparison to the expression of the endogenous WUS2 polypeptide in a control maize plant cell, wherein the endogenous WUS2 polypeptide is encoded by an endogenous polynucleotide that is operably linked to an endogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variant thereof, wherein the at least one exogenous gene transcription agent comprises an artificial zinc finger (AZF) DNA binding domain polypeptide that binds to the DNA sequence of SEQ ID NO:101 in the endogenous maize WUS2 promoter of SEQ ID NO:4, and wherein the maize plant cell can form a regenerable maize plant structure.
 2. The maize plant cell of claim 1, wherein an exogenous polynucleotide encoding a WUS2 polypeptide is absent before and/or during the increase in expression of the endogenous WUS2 polypeptide.
 3. The maize plant cell of claim 1, wherein the increase in expression of the endogenous WUS2 polypeptide is sufficient to increase proliferation, somatic embryogenesis, and/or regeneration capacity of the maize plant cell in comparison to the control maize plant cell.
 4. The maize plant cell of claim 1, wherein the increase in expression of the endogenous WUS2 polypeptide is sufficient to increase transformation efficiency and/or endogenous gene editing efficiency of the maize plant cell in comparison to the control maize plant cell.
 5. The maize plant cell of claim 1, wherein said cell is located within or obtained from a cultured plant tissue explant, an immature embryo, a mature embryo, a leaf, and/or callus or optionally wherein the plant cell is located with or derived from the L1 or L2 layer of the immature or mature embryo.
 6. The maize plant cell of claim 1, wherein the endogenous WUS2 polypeptide comprises an amino acid sequence having at least 95%, 96%, 97%, or 99% amino acid sequence identity across the entire length of SEQ ID NO:2.
 7. A method of producing a regenerable maize plant structure, comprising: (i) introducing into a maize plant cell at least one exogenous gene transcription agent which comprises the polypeptide of SEQ ID NO:93 and transiently increases expression of an endogenous WUS2 polypeptide, wherein the expression is increased in comparison to the expression of the endogenous WUS2 polypeptide in a control maize plant cell, wherein the endogenous WUS2 polypeptide is encoded by an endogenous polynucleotide that is operably linked to an endogenous maize WUS2 promoter of SEQ ID NO:4 or an allelic variant thereof, and wherein the at least one exogenous gene transcription agent comprises an AZF DNA binding domain polypeptide that binds to the DNA sequence of SEQ ID NO:101 in the endogenous maize WUS2 promoter of SEQ ID NO:4; and, (ii) culturing the maize plant cell to produce a regenerable maize plant structure.
 8. The method of claim 7, wherein an exogenous polynucleotide encoding a WUS2 polypeptide is absent before and/or during the increase in expression of the endogenous WUS2 polypeptide.
 9. The method of claim 7, wherein the increase in expression of the endogenous WUS2 polypeptide is sufficient to increase proliferation, somatic embryogenesis, and/or regeneration capacity of the maize plant cell in comparison to the control maize plant cell.
 10. The method of claim 7, wherein the increase in expression of the endogenous WUS2 polypeptide is sufficient to increase transformation efficiency and/or endogenous gene editing efficiency of the maize plant cell in comparison to the control maize plant cell.
 11. The method of claim 7, wherein said cell is located within or obtained from a cultured plant tissue explant, an immature embryo, a mature embryo, a leaf, and/or callus or optionally wherein the plant cell is located with or derived from the L1 or L2 layer of the immature or mature embryo.
 12. The method of claim 7, wherein the endogenous WUS2 polypeptide comprises an amino acid sequence having at least 95%, 96%, 97%, or 99% amino acid sequence identity across the entire length of SEQ ID NO:2. 